Recombinant surfactant protein D compositions and methods of use thereof

ABSTRACT

We describe an rSPD(n/CRD) polypeptide, fragment, homologue, variant or derivative thereof for use in a method of treatment or prophylaxis of a disease. A method of treating an individual suffering from a disease or preventing the occurrence of a disease in an individual is also described, in which the method comprises administering to the individual a therapeutically or prophylactically effective amount of an rSPD (n/CRD) polypeptide, fragment, homologue, variant or derivative thereof. Preferably, the rSPD (n/CRD) polypeptide and nucleic acid comprise SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0001] This application is a continuation-in-part of PCT/GB02/04824,which was filed Oct. 25, 2002 and published in English, whichapplication claimed the priority of Great Britain applications GB0125638.7, filed Oct. 25, 2001, and GB 0209619.6, filed Apr. 26, 2002,the entireties of each of which are hereby incorporated by reference.

[0002] This invention relates to methods of treatment and diagnosis ofdisease, and molecules and compositions for use in such methods.

[0003] We disclose a recombinant fragment of Surfactant Protein D, aswell as a nucleic acid encoding this protein. We demonstrate that thisfragment retains the beneficial properties of Surfactant Protein D, andshow that it may be used to treat various diseases. These diseasesinclude inflammatory diseases, including chronic lung disease, neonatalchronic lung disease, asthma, including allergic asthma, particularlycaused by house dust mite, as well as other diseases. The recombinantSurfactant Protein D fragment we disclose may also be used to treatallergies of various kinds.

SUMMARY

[0004] The present inventors have determined that SP-D binds to freenucleic acid, particularly DNA, via its carbohydrate binding activity.Recognition of free DNA is believed to be responsible for at least someof the beneficial effects of SP-D. Moreover, the inventors havedetermined that the DNA binding properties of SP-D, as well as otherproperties of this molecule, are retained in a recombinant fragmentdesignated rSPD(n/CRD). According to a first aspect of the presentinvention, therefore, we provide an rSPD(n/CRD) polypeptide, fragment,homologue, variant or derivative thereof for use in a method oftreatment or prophylaxis of a disease.

[0005] There is provided, according to a second aspect of the presentinvention, a method of treating an individual suffering from a diseaseor preventing the occurrence of a disease in an individual, the methodcomprising administering to the individual a therapeutically orprophylactically effective amount of an rSPD(n/CRD) polypeptide,fragment, homologue, variant or derivative thereof.

[0006] The disease preferably comprises an inflammatory disease,preferably eczema.

[0007] The disease may comprise an inflammatory disease, preferably aninflammatory lung disease selected from the group consisting of:neonatal chronic lung disease, neonatal respiratory distress syndrome(RDS), adult respiratory distress syndrome, chronic obstructive airwaysdisease (COPD), asthma, cystic fibrosis, pulmonary fibrosis, emphysema,interstitial inflammatory lung disease, sarcoidosis, pneumonia, chronicinflammatory lung disease, neonatal chronic inflammatory lung disease.

[0008] Preferably, the disease is selected from the group consisting of:neonatal chronic lung disease, pulmonary emphysema, chronic obstructivepulmonary disease, cystic fibrosis and asthma.

[0009] The disease may comprise an allergy. The allergy is preferably anallergy is to house dust mite (Dermatophagoides spp), preferablyDermatophagoides pteronyssinus or Dermatophagoides farinae, or to fungior fungal spores, preferably Aspergillus fumigatus. The allergy ispreferably selected from the group consisting of: a seasonal respiratoryallergy, allergic rhinitis, hayfever, nonallergic rhinitis, vasomotorrhinitis, irritant rhinitis, an allergy against grass pollens, treepollens or animal danders, an allergy associated with allergic asthma, afood allergy and allergic eye diseases.

[0010] The disease may be one which is associated with microbialinfection, including bacterial infection and viral infection, preferablya microbial infection of the lung.

[0011] We provide, according to a third aspect of the present invention,a method of reducing airway hyperresponsiveness, serum IgE levels, oreosinopilia in an individual, the method comprising administering to theindividual an rSPD(n/CRD) polypeptide, fragment, homologue, variant orderivative thereof

[0012] As a fourth aspect of the present invention, there is providedmethod of reducing alveolar macrophage number in an individual,preferably by enhancing clearance of apoptotic alveolar macrophages, orby enhancing clearance of necrotic alveolar macrophages, or both, themethod comprising administering to the individual an rSPD(n/CRD)polypeptide, fragment, homologue, variant or derivative thereof.

[0013] In preferred embodiments, the rSPD(n/CRD) polypeptide comprises,preferably consists of, a sequence shown in SEQ ID NO: 1.

[0014] Preferably, a nucleic acid encoding an rSPD(n/CRD) polypeptide,or a fragment, homologue, variant or derivative thereof, preferably anucleic acid comprising a sequence shown in SEQ ID NO: 2, isadministered to the individual.

[0015] We provide, according to a fifth aspect of the present invention,use of a rSPD(n/CRD) polypeptide, nucleic acid, fragment, homologue,variant or derivative thereof in a method of treatment or prophylaxis ofa disease.

[0016] The present invention, in a sixth aspect, provides a recombinantpolypeptide comprising a SP-D fragment having a sequence shown in SEQ IDNO: 1, or a fragment, homologue, variant or derivative thereof.

[0017] In a seventh aspect of the present invention, there is provided anucleic acid comprising a sequence encoding a recombinant polypeptideaccording to the sixth aspect of the invention. Preferably, the nucleicacid comprises the sequence shown in SEQ ID NO: 2.

[0018] According to an eighth aspect of the present invention, weprovide a vector, preferably an expression vector, comprising a nucleicacid sequence according to the seventh aspect of the invention.

[0019] We provide, according to a ninth aspect of the invention, a hostcell transformed with a vector according to the eighth aspect of theinvention. There is provided, in accordance with a tenth aspect of thepresent invention, a pharmaceutical composition comprising a recombinantpolypeptide according to the sixth aspect of the invention, a nucleicacid according to the seventh aspect of the invention, a vectorcomprising a nucleic acid according to the eighth aspect of theinvention, or a host cell according to the ninth aspect of theinvention, together with a pharmaceutically acceptable carrier ordiluent.

[0020] As an eleventh aspect of the invention, we provide a method ofidentifying a molecule which binds to an rSPD(n/CRD) polypeptide, themethod comprising exposing a rSPD(n/CRD) polypeptide, fragment,homologue, variant or derivative thereof to a candidate molecule anddetecting whether the candidate molecule binds to the rSPD(n/CRD)polypeptide, etc.

[0021] According to a twelfth aspect of the present invention, weprovide a method of identifying an agonist or antagonist of anrSPD(n/CRD) polypeptide, the method comprising: (a) providing a cell ororganism; (b) exposing the cell or organism to an rSPD(n/CRD)polypeptide, nucleic acid, or a fragment, homologue, variant orderivative thereof; (c) exposing the cell to a candidate molecule; and(d) detecting an rSPD(n/CRD) mediated effect.

[0022] The rSPD(n/CRD) mediated effect is preferably selected from thegroup consisting of: reduction of peripheral blood eosinophilia,reduction of serum IgE levels, reduction of serum IgG1 levels, reductionin airway hyperresponsiveness, reduction in alveolar macrophage number,reduction of phospholipid levels in lavage, down-regulation of Eotaxinexpression, reduction in MCP-1 expression, down-regulation of MIP-1αexpression, and down-regulation of MTP-2 expression. The methodpreferably further comprises isolating or synthesising a selected oridentified molecule.

[0023] There is provided, according to a thirteenth aspect of thepresent invention, a molecule identified or selected using a methodaccording to the twelfth aspect of the invention.

[0024] We provide, according to a fourteenth aspect of the presentinvention, composition comprising an rSPD(n/CRD) polypeptide, nucleicacid, or a fragment, homologue, variant or derivative thereof, togetherwith one or more of the following components: a lung extract, preferablya bovine, preferably, calf lung extract, or a component thereof aphospholipid, preferably a phosphatidylcholine or a disaturatedphosphatidylcholine, a fatty acid, a surfactant-associated protein,preferably a surfactant-associated protein B or a surfactant-associatedprotein C, a cetyl alcohol, a long-chain repeating alcohol, tyloxapol,colfosceril palmitate (dipalmitoylphosphatidylcholine), palmitic acid,tripalmitin, poractant alfa and sodium chloride.

[0025] As a fifteenth aspect of the present invention, there is provideduse of an rSPD(n/CRD) polypeptide, nucleic acid, or a fragment,homologue, variant or derivative thereof in surfactant replacementtherapy.

[0026] We provide, according to a sixteenth aspect of the presentinvention, an rSPD(n/CRD) polypeptide, nucleic acid, or a fragment,homologue, variant or derivative thereof in surfactant replacementtherapy for use in surfactant replacement therapy.

[0027] According to a seventeenth aspect, there is provided the use ofSP-D or a fragment thereof in the manufacture of a composition for thetreatment of a condition involving the production of free DNA on thesurface of cells. SP-D and fragments thereof, including rSPD(n/CRD),bind to DNA via their carbohydrate binding capacity. The fragments ofSP-D accordingly advantageously retain the carbohydrate binding abilityof native SP-D. The invention moreover provides a method for treating apatient suffering from a condition involving the production of free DNAon the surface of cells comprising administering to a patient in need ofsuch treatment a pharmaceutically effective amount of SP-D or a fragmentthereof.

[0028] The invention provides the use of SP-D or a fragment thereof inthe manufacture of a composition for the treatment of a chronicinfection which results in the accumulation of free nucleic acid in thelung. For example, the infection may be a bacterial, viral or fungalinfection.

[0029] The invention moreover provides the use of SP-D or a fragmentthereof in the manufacture of a composition for the treatment of cysticfibrosis. In cystic fibrosis, free nucleic acid is produced by bacterialcells in the lung, which leads to bacterial bio-film formation andpersistent bacterial lung infection. SP-D and fragments thereof also actto opsonise many pulmonary pathogens, increasing the effectiveness ofcystic fibrosis treatment.

[0030] In a further aspect, there is provided the use of SP-D or afragment thereof in the manufacture of a composition for the clearanceof necrotic and apoptotic cells. Such cells have DNA at their surfaces,which is bound by SP-D and fragments thereof for phagocyte-mediatedclearance. Thus, the invention extends to compositions for the treatmentof lung diseases and conditions such as COPD, chronic inflammation andchronic asthma using SP-D or fragments thereof and to methods fortreating a patient suffering from a selected from the group consistingof cystic fibrosis, lung diseases, COPD, chronic inflammation, chronicbacterial infection, chronic viral infection, chronic fungal infectionand chronic asthma comprising administering to a patient in need of suchtreatment a pharmaceutically effective amount of SP-D or a fragmentthereof.

[0031] In a still further aspect, there is provided the use of SP-D or afragment thereof in the manufacture of a composition for the treatmentof autoantibody production in autoimmune diseases; as well as a methodfor treating a patient suffering from an autoimmune disease comprisingadministering to a patient in need of such treatment a pharmaceuticallyeffective amount of SP-D or a fragment thereof.

[0032] Free DNA or DNA debris from necrotic or apoptotic cells can actas an autoantigen, resulting in auto antibody production. Clearance ofsuch free DNA by SP-D and fragments thereof reduces autoantibodyproduction. Other aspects of the invention are set out in theindependent and dependent claims, as well as set out in the description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033]FIG. 1A depicts the structure a recombinant fragment of human SP-D(rSPD(n/CRD)). This is composed of trimers of the neck region andcarbohydrate recognition domain (CRD) and has been expressed, purifiedand characterised in the MRC Immunochemistry Unit at Oxford University.

[0034]FIG. 1B. SDS-PAGE of 1 week culture filtrate of Aspergillusfumigatus showing a dominant 18 kDa band (Asp f 1).

[0035]FIG. 2A. Serum IgE in Afu mouse model measured 3 days aftertreatment with 5 daily doses of 10 μg rSPD(n/CRD) or PBS givenintranasally after intranasal challenge with Afu 1wcf and afterre-challenge 1 week later with Afu alone. P=PBS treated nonsensitisedmice, AP=PBS treated sensitised mice, AR=rSPD(n/CRD) treated sensitisedmice, APrc=Afu rechallenged mice treated with PBS, ARrc=Afu rechallengedmice treated with rSPD(n/CRD).

[0036]FIG. 2B. Afu-specific IgE in Afu mouse model. As FIG. 2A above,but Afu-specific IgG1 is measured.

[0037]FIG. 3. Peripheral blood eosinophilia in Afu mouse model measured1 day after treatment with 4 daily doses of 10 μg rSPD(n/CRD) or PBSgiven intranasally after intranasal challenge with Afu 1wcf and afterre-challenge 1 week later with Afu alone. P=PBS treated non-sensitisedmice, AP=PBS treated sensitised mice, AR=rSPD(n/CRD) treated sensitisedmice, APrc=Afu rechallenged mice treated with PBS, ARrc=Afu rechallengedmice treated with rSPD(n/CRD).

[0038]FIGS. 4A, 4B and 4C. Cytokines in Afu mouse model measured 1 dayafter treatment with 2 daily doses of 10 μg rSPD(n/CRD) givenintranasally alter intranasal challenge with Afu 1wcf. FIG. 4A IL-12measured in the spleen, FIG. 4B IFN-γ measured in the spleen and FIG. 4CIL-4 measured in the spleen. P=PBS treated nonsensitised mice, AP=PBStreated sensitised mice, AR=rSPD(n/CRD) treated sensitised mice,AA=rhSP-A treated sensitised mice.

[0039]FIGS. 5A and 5B. Airway hyper-responsiveness in Afu mouse model.Mice are treated with 4 daily doses of 10 μg rSPD(n/CRD) givenintranasally after intranasal challenge with Afu 1wcf. Plethysmographyis measured 2 hours after an intranasal rechallenge with Afu 1wcf alonegiven 3 days after completion of treatment. Figures A Dose response toincreasing doses of methacholine. FIG. 5B Response to 30 mg/mlmethacholine after treatment with 4 daily doses of 10 μg rSPD(n/CRD) orrhSP-A given intranasally after intranasal challenge with Afu 1wcf.P=PBS treated non-sensitised mice, AP=PBS treated sensitised mice,AR=rSPD(n/CRD) treated sensitised mice, AA=rfhSP-A treated sensitisedmice.

[0040]FIGS. 6A and 6B. Afu mouse model H&E stained lung sections ofsensitised mice after treatment FIG. 6A with PBS FIG. 6B withrSPD(n/CRD) or c nonsensitised mouse treated with PBS.

[0041]FIG. 7A. An analysis of IL-12 in the lung homogenates of Der psensitised mice after treatment measured by intracellular cytokinestaining followed by FACS analysis for the percentage of highly stainedcells (PE>1000) positive for IL-12. PBS non-sensitised mice treated withPBS. HP=sensitised mice treated with PBS. HR=sensitised mice treatedwith 10 μg rSP-D (N/CRD).

[0042]FIG. 7B. An analysis of IL-12 in the spleen homogenates of Der psensitised mice after treatment measured by intracellular cytokinestaining followed by FACS analysis for the percentage of highly stainedcells (PE>1000) positive for IL-12. PBS=non-sensitised mice treated withPBS. HP=sensitised mice treated with PBS. ER=sensitised mice treatedwith 10 μg rSP-D (N/CRD). HB=sensitised mice treated with 10 μg BSA.

[0043]FIG. 7C. An analysis of IFN-γ in the spleen homogenates of Der psensitised mice after treatment measured by intracellular cytokinestaining followed by FACS analysis for the percentage of highly stainedcells (PE>1000) positive for IFN-γ. PBS=non-sensitised mice treated withPBS. HP=sensitised mice treated with PBS. HR=sensitised mice treatedwith 10 μg rSP-D (N/CRD).

[0044]FIG. 7D. An analysis of TNF-α in the spleen homogenates of Der psensitised mice after treatment measured by intracellular cytokinestaining followed by FACS analysis for the percentage of highly stainedcells (PE>1000) positive for TNF-α. PBS=non-sensitised mice treated withPBS. HP=sensitised mice treated with PBS. HR=sensitised mice treatedwith 10 μg rSP-D (N/CRD).

[0045]FIG. 8A (entitled “Average Penh response to a 1 min challenge with20 mg/ml methacholine on the 4th day of treatment”) is a graph showingthe effect of treatment with rSPD(n/CRD) on the airwayhyperresponsiveness (AHR) of allergic mice, measured on the last day of4 days of treatment. The allergic mice are sensitised toDermatophagoides pteronyssinus. The PBS control representsnon-sensitised mice treated with PBS. Average Perth response to a 1minute challenge with 20 mg/ml methacholine on the 4th day of treatmentis shown. X-axis: left: PBS control, middle: Der p-PBS, right: Derp-rSPD; Y-axis: % Penh above background.

[0046]FIG. 8B (entitled “Airway hyperresponsiveness in allergic miceinduced by methacholine challenge”) is a graph showing the dose responseto methacholine, following allergen re-challenge 4 days after treatmentwith PBS or rSPD(n/CRD). The allergic mice are sensitised toDermatophagoides pteronyssinus. The PBS-PBS control representsnon-sensitised mice treated with PBS. Airway hyperresponsiveness (AHR)is measured in allergic mice induced by methacholine challenge. X-axis:methacholine (mg/ml); diamonds: PBS-PBS, squares: Der p-PBS, triangles:Der p-rSPD.

[0047]FIG. 9 is a graph showing the effect of rSP-D (N/CRD) treatment onalveolar macrophage number, following lavage. X-axis: age in weeks;Y-axis: alveolar macrophage number×10⁴ per mouse lavage.

[0048]FIG. 10A shows a cytospin of cells in bronchoalveolar lavage.Alveolar macrophages are stained with Malachite green (upper panels) orcrystal violet (lower panels) after cytospin. Morphology is shown for notreatment (upper left panels), BSA treated (upper right panels) andr-SP-D treated (lower panels).

[0049]FIG. 10B shows a cytospin of cells in bronchoalveolar lavage.Upper left: untreated cells from SP-D deficient mice; upper right: cellsfrom wild-type mice; lower left: cells from SP-D deficient mice treatedwith BSA; lower right: cells from SP-D deficient mice treated withrSP-D.

[0050]FIG. 11 (entitled “Phospholipid in cell-free lavage supernatantper mouse”) is a graph showing the effect of rSP-D (N/CRD) treatment ontotal BAL phospholipid levels. Phospholipid in cell-free lavagesupernatant is shown per mouse. X-axis: age in weeks; Y-axis:phospholipid in μg per mouse.

[0051]FIG. 12 (entitled “Chemokine mRNA levels in SP-D KO mice comparedto WT and the effect of treatment with rSP-D”) are graphs showingchemokine mRNA levels in SP-D knockout mice compared to wild type andthe effect of treatment with rSP-D(N/CRD).

[0052]FIG. 13A shows a representative result of forward scatter (cellsize) and side scatter (granularity) flow cytometry in SP-D deficientmice compared to wild-type mice.

[0053]FIG. 13B shows the typical patterns of annexin V and PI stainingof macrophages from SP-D deficient mice compared to wild-type mice.

[0054]FIG. 13C shows the extent of co-labelling of FITC-labelled rSP-Dwith annexin V and/or PI positive cells.

[0055]FIG. 13D is a graph showing percentage apoptotic cells (upperpanel) and necrotic cells (lower panel) in bronchoalveolar lavage ofuntreated SP-D knock-out mice, wild-type mice, rSPD(n/CRD) treated mice,PBS treated mice, SP-A treated mice and BSA treated mice.

[0056]FIG. 14 is a graph showing GM-CSF concentrations inbronchoalveolar lavage of wild-type, SP-D knock-out mice and SP-Dknock-out mice treated with rSPD(n/CRD).

[0057]FIG. 15 shows confocal microscopy of macrophages from wild-type(panels A and B) and knock-out mice (panels C and D), stained withFITC-dUTP (green). Cells in advanced apoptosis stain green and cells inearly apoptosis are identified by characteristic punctate staining ofend labelled DNA fragments.

[0058]FIG. 16 shows enhanced phagocytosis of apoptotic (orange labelled)macrophages by freshly isolated green macrophages.

[0059]FIG. 17 shows plasma cholesterol levels in mM in wild-type andSP-D knock-out mice.

[0060]FIG. 18 shows negatively stained transmission electron microscopicimages of genomic DNA and complexes between SP-D and DNA. A, genomicDNA; B & C, genomic DNA complexed with rSPD(n/CRD); D, genomic DNAcomplexed with native SP-D.

[0061]FIG. 19 demonstrates binding between rSPD(n/CRD) and mannan iscompeted by deoxyribonucleotides. A fixed concentration of rSPD(n/CRD)(100 μg/mL) was allowed to bind to biotinylated mannan that wasimmobilised on a SA BIAcore chip in the presence of indicatedconcentrations of hexoses (A) or dNTPs (B) in 5 mM CaCl₂-containingsaline buffer.

[0062]FIG. 20 shows inhibition of propidum iodide binding byrSPD(n/CRD).

[0063]FIG. 21 shows that alveolar macrophages isolated from SP-Dknock-out mouse are defective in clearing DNA. Alveolar macrophages wereisolated from the lung lavage of wild-type (A,D) or SP-A knock-out mice(SP-A^((−/−)))(B,E) or SP-D knockout mice (SP-D^((−/−)))(C,F), washedand incubated with either buffer (A,B,C) or Cy3-labelled (red) lung DNA(D,E,F). The uptake of DNA was monitored by FACS at FL2-H and graphedagainst a non-specific FL1-H. The proportion of alveolar macrophagesthat took up the DNA was indicated in the figure (D,E,F).

DETAILED DESCRIPTION

[0064] We disclose the sequence and method of production of arecombinant SP-D fragment, referred to here as “rSPD(n/CRD)polypeptide”. As far as we are aware, the sequence and production ofrSPD(n/CRD) polypeptide has not previously been made known. We show thatrSPD(n/CRD) polypeptides are capable of associating with each other andforming timers in solution.

[0065] In a highly preferred embodiment, we describe the use ofrSPD(n/CRD) polypeptide, nucleic acid, or a fragment, homologue, variantor derivative thereof for use as a surfactant replacement therapy,particularly for neonates. rSPD(n/CRD) polypeptide, nucleic acid, or afragment, homologue, variant or derivative thereof may suitably beemployed for the treatment of neonatal lung diseases, particularly,neonatal chronic lung diseases.

[0066] We demonstrate that rSPD(n/CRD) polypeptide reduces peripheralblood eosinophilia and total serum IgE levels, as well as IgG1 levels,when administered to knock-out mice lacking Surfactant Protein D andchallenged with allergen. rSPD(n/CRD) polypeptide is therefore suitablefor use as a modulator of allergic hypersensitivity reactions inindividuals. rSPD(n/CRD) polypeptide is also suitable for use intreating any disease or syndrome characterised in peripheral bloodeosinophilia, elevated serum IgE levels, elevated IgG1 levels, or anycombination of these.

[0067] rSPD(n/CRD) polypeptide may also be used for regulation ofmacrophage mediated inflammation in the lung, as well as in the defenceagainst invasion by pathogens and in modulating inflammatory responsesto infection and allergenic stimuli.

[0068] Mice, for example C57B116 wild-type mice, sensitised andchallenged with house dust mite allergen show airway hyperresponsivenesscharacteristic of allergic asthma. Administration of rSPD(n/CRD)polypeptide to such mice reduces airway hyperresponsiveness.Accordingly, rSPD(n/CRD) polypeptide may be administered to individualsto treat any disease or syndrome in which airway hyperresponsivenessoccurs, including allergic asthma.

[0069] Other diseases for which treatment of rSPD(n/CRD) polypeptide maybe employed include skin conditions, for example, eczema (which is a Th2mediated disease), as well as allergies in general. Such allergiesinclude food allergies, for example against allergens such as egg, treenut, peanut, and milk. Other common allergens include pollens, dust,mould, and mildew, as well as insect, domestic animal (dog, cat, bird),and plant allergens. Also included are seasonal respiratory allergies,commonly referred to as hay fever, aeroallergens, which include housedust mite, fungal spores, grass pollens, tree pollens and animaldanders.

[0070] Other categories of allergies, which may be treated withrSPD(n/CRD) are those which might benefit from the reduction in airwayhyperresponsiveness, serum IgE and eosinophilia which are major factorsin producing the symptoms of allergy. This includes the treatment ofallergic asthma. rSPD(n/CRD) may also be used as an effective treatmentfor conditions that would benefit from the up-regulation of thecell-mediated immune system. This includes anti-microbial action and thetreatment of lung infections.

[0071] rSPD(n/CRD) may be used as an effective treatment for conditionsthat would benefit from the up-regulation of the activity of NaturalKiller cells and the secretion of the cytokine IFNγ by cells of theimmune system. This includes the treatment of lung cancer and othercancers and neoplasms.

[0072] rSPD(n/CRD) may also be used as an effective treatment forconditions that would benefit from the supplementation of endogenousnatural SP-D with rSPD(n/CRD). This includes the treatment of conditionsin which the level of endogenous natural SP-D is abnormally low. Thisincludes the treatment of neonates with disorders of the lung surfactantsystem in which the level of endogenous natural SP-D is abnormally low.This also includes the treatment of cystic fibrosis patients in whichthe level of endogenous natural lung SP-D is abnormally low. This alsoincludes the treatment of patients with other infections of the lung inwhich the level of endogenous natural SP-D is abnormally low.

[0073] In a further embodiment, SP-D's anti-allergic properties can beharnessed to combat allergic eye disease. The invention thus providesSP-D, or a fragment thereof such as rSPD(n/CRD), for the treatment ofallergic eye conditions.

[0074] We have measured surfactant protein D levels in human tears andfound this to be 132 ng/ml. The fact that SP-D has anti-allergicproperties as set forth herein and is present in human tear fluidsuggests that the protein is involved in the natural anti-allergiceffect of tears. SP-D or rSP-D(n/CRD) can be added to, for example,contact lens solution to help offset irritation/inflammation by contactlenses. Moreover, it is a valuable addition in the treatment of forexample Sjogren's syndrome—an autoimmune disease characterised by dryeyes dry mouth (caused by autoantibodies to human salivary and lacrimalgland components). This is currently treated with artificial tearsupplementation (such as hypermellose eye drops) and the addition ofSP-D or fragments thereof to such formulations enhances the protectiveeffect of artificial tears against eye infections and cornealinflammation.

[0075] Where reference is made to rSPD(n/CRD) above, this should betaken to refer to the polypeptide itself or a fragment, homologue,variant or derivative thereof.

[0076] Where reference is made to a treatment of a disease, this shouldbe taken to include reference to alleviation of a symptom of thatdisease. Preferably, substantially all of the symptoms of an individualhaving that disease are alleviated or removed. “Disease” should be takento include any syndrome, as well as any condition affecting the healthor well-being of an individual. Preferably, an individual to which anrSPD(n/CRD) polypeptide, fragment, homologue, variant or derivativethereof is administered is relieved to at least one symptom of thedisease or condition, i.e., he reverts substantially to the state of anormal unaffected individual. This may be assessed by a physician usinga relevant clinical parameter. For example, airway hyperresponsivenessmay be measured using the methods set out in the Examples.

[0077] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of chemistry, molecularbiology, microbiology, recombinant DNA and immunology, which are withinthe capabilities of a person of ordinary skill in the art. Suchtechniques are explained in the literature. See, for example, Sambrook,J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: ALaboratory Manual, Second Edition, Books 1-3, Cold Spring HarborLaboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements;Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley &Sons, New York, N.Y.); Roe, B., J. Crabtree, and A. Kahn, 1996, DNAIsolation and Sequencing: Essential Techniques, John Wiley & Sons;Polak, J. M. and James O'D. McGee, 1990, In Situ Hybridization:Principles and Practice; Oxford University Press; M. J. Gait (Editor),1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press; and,Lilley, D. M. J. and J. E. Dahlberg, 1992, Methods of Enzymology: DNAStructure Part A: Synthesis and Physical Analysis of DNA, Methods inEnzymology, Academic Press. Each of these general texts is hereinincorporated by reference.

[0078] Surfactant Protein D rSPD(n/CRD)

[0079] Surfactant Protein D has been identified and characterisedpreviously, in for example Rust et al., 1991, Human surfactant proteinD: SP-D contains a C-type lectin carbohydrate recognition domain,Archives of Biochemistry and Biophysics 290 (1):116-126; Lu et al.,1992, Purification, Characterization and cDNA Cloning of Human LungSurfactant Protein D, Biochem. J. 284:785-802; Crouch et al., 1993,Genomic organization of human surfactant protein D (SP-D). SP-D isencoded on chromosome 10q22.2-23.1, The Journal of Biological Chemistry268 (4):2976-2983; Kolble et al, 1993, Assignment of the human pulmonarysurfactant protein D gene (SFTP4) to 10q22-q23 close to the surfactantprotein A gene cluster, Genomics 17(2):294-298.

[0080] Where the terms “Surfactant Protein D”, “SP-D”, “hSP-D” and“natural SP-D” are used, these should be taken to refer to anySurfactant Protein D polypeptide or nucleic acid (as the contextrequires). Preferably, however, these terms should be taken to refer tohuman Surfactant Protein-D, for example, the sequences disclosed in theabove references, or in GenBank accession numbers NM_(—)003019.1,XM_(—)005776.2, X65018.1 and L05485.1. Most preferably, the SurfactantProtein D is a human Surfactant Protein D having the GenBank accessionnumbers NM_(—)003019.1. The nucleic acid and amino acid sequences ofsuch a human SP-D are shown in SEQ ID NO: 3 and SEQ ID NO: 4respectively.

[0081] Recombinant Surfactant Protein D rSPD(n/CRD) Polypeptides

[0082] We provide for a polypeptide fragment of Surfactant Protein D(SP-D), which we refer to as rSPD(n/CRD). The sequence of such arSPD(n/CRD) polypeptide is shown in SEQ ID NO: 1. rSP-D (N/CRD) is alsoreferred to in this document as “rhSP-D”, and the two terms should beregarded as synonymous.

[0083] Where the term rSP-D is employed, this should be taken to refergenerally to recombinant SP-D, whether corresponding to full lengthSP-D, preferably human SP-D (expressed in a recombinant manner), or afragment of this. Preferably, the term rSP-D should be taken to refer toa recombinant fragment of SP-D, preferably human SP-D. Preferably, andwhere the context requires, “rSP-D” should be taken to refer to therecombinant SP-D fragment rSPD(n/CRD) described above.

[0084] It will be understood that polypeptides disclosed here are notlimited to the particular rSPD(n/CRD) sequence shown in SEQ ID NO: 1,but also include fragments thereof. Particularly preferred fragmentsinclude those having one or more biological activities of rSPD(n/CRD).

[0085] Furthermore, the rSPD(n/CRD) polypeptides also generally includeany recombinant fragment of SP-D, preferably human SP-D, which lacks theN-terminal domain and/or the collagen domain, preferably both. Thus, ina preferred embodiment, the rSPD(n/CRD) polypeptide is a recombinantfragment of SP-D, preferably human SP-D depicted in SEQ ID NO: 4, whichlacks substantially lacks residues 1-178. In a further preferredembodiment, the rSPD(n/CRD) polypeptide is a recombinant fragment ofSP-D, preferably human SP-D sequence shown in SEQ ID NO: 4, comprisingsubstantially residues 179-355.

[0086] In a preferred embodiment, the proline residue corresponding toposition 200 of the human SP-D sequence (SEQ ID NO: 4) is replaced byanother residue. Preferably, the proline residue is replaced with auncharged polar residue, for example, a cysteine, serine, threonine ormethionine residue. In a highly preferred embodiment, the prolineresidue is replaced with a serine residue. Thus, the rSPD(n/CRD)polypeptide may comprise a sequence shown in SEQ ID NO: 1.

[0087] Fragments, homologues, variants and derivatives of each of theabove sequences are also included.

[0088] In preferred embodiments, the rSPD(n/CRD) polypeptide comprises a“head” region or carbohydrate recognition domain (CRD), comprisingsubstantially the following residues:VELFPNGQSVGEKIFKTAGFVKPFTEAQLLCTQAGGQLASPRSAAENAALQQLVVAKNEAAFLSMTDSKTEGKFTYPTGESLVYSNWAPGEPNDDGGSEDCVEIFTNGKWNDRACGEKRLVVCEF

[0089] Preferably, the rSPD(n/CRD) polypeptide comprises means formultimerisation, preferably trimerisation, with another rSPD(n/CRD)polypeptide. Such means may include for example, a biotin moiety whichinteracts with and binds to an avidin or streptavidin moiety on anotherrSPD(n/CRD) polypeptide.

[0090] In further preferred embodiments, the rSPD(n/CRD) polypeptidefurther comprises a “neck” region comprising substantially the followingresidues:

[0091] DVASLRQQVEALQGQVQHLQAAFSQYKK

[0092] Preferably, such a neck region is N terminal to the carbohydraterecognition domain (CRD).

[0093] In a preferred embodiment, the rSPD(n/CRD) polypeptide furthercomprises at least one Gly-Xaa-Yaa stretch, preferably a sequencecomprising a plurality of Gly-Xaa-Yaa repeats, most preferably asequence comprising 8 Gly-Xaa-Yaa repeats. In a preferred embodiment,the rSPD(n/CRD) polypeptide further comprises a N-terminal sequencecomprising substantially GSPGLKGDKGIPGDKGAKGESGLP.

[0094] In a highly preferred embodiment, the rSPD(n/CRD) polypeptidecomprises, preferably consists of, a sequence shown in SEQ ID NO: 1.Thus, as used in this document, the term “rSPD(n/CRD) polypeptide”should preferably be taken to refer to the sequence below:GSPGLKGDKGIPGDKGAKGESGL PDVASLRQQVEAL (SEQ ID NO: 1)QGQVQHLQAAFSQYKKVELFPNGQSVGEKIFKTAGFVKPFTEAQLLCTQAGGQLASPRSAAENAALQQLVVAKNEAAFLSMTDSKTEGKFTYPTGESLVYSNWAPGEP NDDGGSEDCVEIFTNGKWNDRACGEKRLVVCEF,

[0095] as well as a fragment, homologue, variant or derivative thereof.A (G-Xaa-Yaa)₈ motif is present at amino acids 1-24, a “neck” domain ispresent at amino acids 25-53, and a “head” domain is present at aminoacids 54-177. Nucleotide sequences of SEQ ID NO:2 corresponding to thesedomains are: (G-Xaa-Yaa)₈, 1-72; “neck,” 73-156; and “head,” 157-534 .

[0096] The polypeptides disclosed also include homologous sequencesobtained from any source, for example related viral/bacterial proteins,cellular homologues and synthetic peptides, as well as variants orderivatives thereof. Thus polypeptides also include those encodinghomologues of rSPD(n/CRD) from other species including animals such asmammals (e.g. mice, rats or rabbits), especially primates, moreespecially humans. More specifically, homologues include humanhomologues.

[0097] Thus, we disclose variants, homologues or derivatives of theamino acid sequence of rSPD(n/CRD) sequence shown in SEQ ID NO: 1, aswell as variants, homologues or derivatives of a nucleotide sequenceencoding such amino acid sequences.

[0098] Preferably, the rSPD(n/CRD) polypeptides, variants, homologues,fragments and derivatives disclosed here comprise one or more propertiesof rSPD(n/CRD), preferably one or more biological activities ofrSPD(n/CRD). Thus, the variants, etc. preferably comprise one or moreactivities including but not limited to, carbohydrate binding activity,multimerisation activity, including trimerisation activity,down-regulation of chemokines, reduction of airway hyperresponsiveness,reduction in alveolar macrophage number, reduction in phospholipidlevel, when administered to an animal lacking SP-D, increased clearanceof apoptotic and/or necrotic macrophages, reduction in peripheral bloodeosinophilia, reduction in serum IgE, reduction in serum IgG1, as wellas any of the biological activities or properties disclosed in theExamples.

[0099] In the context of this document, a homologous sequence is takento include an amino acid sequence which is at least 15, 20, 25, 30, 40,50, 60, 70, 80 or 90% identical, preferably at least 95 or 98% identicalat the amino acid level over at least 50 or 100, preferably 200, 300,400or 500 amino acids with the sequence of rSPD(n/CRD) shown in SEQ IDNO: 1. In particular, homology should typically be considered withrespect to those regions of the sequence known to be essential forprotein function rather than nonessential neighbouring sequences. Thisis especially important when considering homologous sequences fromdistantly related organisms.

[0100] Although homology can also be considered in terms of similarity(i.e. amino acid residues having similar chemical properties/functions),in the context of the present invention it is preferred to expresshomology in terms of sequence identity.

[0101] Homology comparisons can be conducted by eye, or more usually,with the aid of readily available sequence comparison programs. Thesepublicly and commercially available computer programs can calculate %homology between two or more sequences.

[0102] % homology may be calculated over contiguous sequences, i.e. onesequence is aligned with the other sequence and each amino acid in onesequence directly compared with the corresponding amino acid in theother sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues (for example less than 50 contiguousamino acids).

[0103] Although this is a very simple and consistent method, it fails totake into consideration that, for example, in an otherwise identicalpair of sequences, one insertion or deletion will cause the followingamino acid residues to be put out of alignment, thus potentiallyresulting in a large reduction in % homology when a global alignment isperformed. Consequently, most sequence comparison methods are designedto produce optimal alignments that take into consideration possibleinsertions and deletions without penalising unduly the overall homologyscore. This is achieved by inserting “gaps” in the sequence alignment totry to maximise local homology.

[0104] However, these more complex methods assign “gap penalties” toeach gap that occurs in the alignment so that, for the same number ofidentical amino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons. For example when using the GCG Wisconsin Bestfitpackage (see below) the default gap penalty for amino acid sequences is−12 for a gap and −4 for each extension.

[0105] Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (University of Wisconsin,U.S.A; Devereux et al., 1984, Nucleic Acids Research 12:387). Examplesof other software than can perform sequence comparisons include, but arenot limited to, the BLAST package (see Ausubel et al., 1999 ibid—Chapter18), FASTA (Atschul et al., 1990, J. Mol. Biol. 403-410) and theGENEWORKS suite of comparison tools. Both BLAST and FASTA are availablefor offline and online searching (see Ausubel et al., 1999 ibid., pages7-58 to 7-60). However it is preferred to use the GCG Bestfit program.

[0106] Although the final % homology can be measured in terms ofidentity, the alignment process itself is typically not based on anall-or-nothing pair comparison. Instead, a scaled similarity scorematrix is generally used that assigns scores to each pairwise comparisonbased on chemical similarity or evolutionary distance. An example ofsuch a matrix commonly used is the BLOSUM62 matrix—the default matrixfor the BLAST suite of programs. GCG Wisconsin programs generally useeither the public default values or a custom symbol comparison table ifsupplied (see user manual for further details). It is preferred to usethe public default values for the GCG package, or in the case of othersoftware, the default matrix, such as BLOSUM62.

[0107] Once the software has produced an optimal alignment, it ispossible to calculate % homology, preferably % sequence identity. Thesoftware typically does this as part of the sequence comparison andgenerates a numerical result.

[0108] The terms “variant” or “derivative” in relation to the amino acidsequences of the present invention includes any substitution of,variation of, modification of, replacement of, deletion of or additionof one (or more) amino acids from or to the sequence providing theresultant amino acid sequence retains substantially the same activity asthe unmodified sequence, preferably having at least the same activity asthe rSPD(n/CRD) polypeptide shown in SEQ ID NO: 1.

[0109] Polypeptides having the amino acid sequence shown in theExamples, or fragments or homologues thereof may be modified for use inthe methods and compositions described here. Typically, modificationsare made that maintain the biological activity of the sequence. Aminoacid substitutions may be made, for example from 1, 2 or 3 to 10, 20 or30 substitutions provided that the modified sequence retains thebiological activity of the unmodified sequence. Alternatively,modifications may be made to deliberately inactivate one or morefunctional domains of the polypeptides described here. Functionaldomains of rSPD(n/CRD) include the collagen domain, the neck region andthe carbohydrate recognition domain. Amino acid substitutions mayinclude the use of non-naturally occurring analogues, for example toincrease blood plasma half-life of a therapeutically administeredpolypeptide.

[0110] Conservative substitutions may be made, for example according tothe Table below. Amino acids in the same block in the second column andpreferably in the same line in the third column may be substituted foreach other: ALIPHATIC Non-polar G A P I L V Polar-uncharged C S T M N QPolar-charged D E K R AROMATIC H F W Y

[0111] As an example, relative to SEQ ID NO: 1, each of thesubstitutions G59A, I66L, F76Y, E78D and Q80N represent conservativesubstitutions.

[0112] Polypeptides also include fragments of the full length sequenceof Surfactant Protein D. Preferably fragments comprise at least oneepitope. Methods of identifying epitopes are well known in the art.Fragments will typically comprise at least 6 amino acids, morepreferably at least 10, 20, 30, 50 or 100 amino acids.

[0113] Polypeptides also include fragments of the full length sequenceof Surfactant Protein D (SEQ ID NO: 4) or rSPD(n/CRD) (SEQ ID NO: 1)that retain one or more biological activities of the polypeptide of SEQID NO: 4 or SEQ ID NO: 1. Preferably fragments comprise at least oneepitope. Methods of identifying epitopes are well known in the art. Anepitope will typically comprise at least 6 amino acids, more preferablyat least 10, 20, 30, 50, or 100 amino acids.

[0114] As used herein, the phrase “retain one or more biologicalactivities” means that a fragment has at least 20% or one or morebiological activities of the full length rSPD(n/CRD) of SEQ ID NO: 1.

[0115] Fragments of rSPD(n/CRD) include, for example, polypeptidescomprising amino acids 4-177, 7-177, 10-177, 13-177, 16-177, 19-177,22-177, 25-177, 30-177, 35-177, 40-177, 45-177, 50-177, 53-177, 60-177,70-177, 80-177, 90-177, 100-177, 110-177, 120-177, 130-177, 140-177,1-176, 1-175, 1-174, 1-173, 1-172, 1-171, 1-170, 1-160, 1-150, 1-140,1-130, 1-120, 1-110, 1-100, 1-90, 1-80, 1-70, 1-60, 25-176, 25-175,25-174, 25-173, 25-172, 25-171, 25-170, 25-160, 25-150, 25-140, 25-130,25-120, 25-110, 25-100, 25-90, 25-80, 25-70, 54-177, 54-176, 54-175,54-174, 54-173, 54-172, 54-170, 54-160, 54-150, 54-140, 54-130, 54-120,54-110, 54-100, 54-90, 54-80 and 54-70 of SEQ ID NO: 1, andcorresponding fragments of SEQ ID NO: 4. The above-noted fragments andothers are encoded by the corresponding nucleic acid fragment sequencesfound within SEQ ID NO: 2 and/or SEQ ID NO: 3.

[0116] As used herein, the terms “reducing,” “reduces,” “decreasing,”“decreases,” “down-regulating” or “down-regulation,” when applied to asymptom of a disease or disorder or to a measurable parameter, e.g.,alveolar macrophage number, expression of a gene, etc., means at least a10% reduction relative to the symptom or parameter measured in a system,model or individual not administered an rSPD(n/CRD) polypeptide.

[0117] As used herein, the terms “enhances,” “enhancing,” “increases” or“increasing,” when applied to a measurable parameter, e.g., apoptotic ornecrotic macrophage clearance, etc., means at least a 10% reductionrelative to the parameter measured in a system, model or individual notadministered an rSPD(n/CRD) polypeptide.

[0118] Recombinant SP-D, including r-SP-D(N/CRD) and its fragments,homologues, variants and derivatives, are typically made by recombinantmeans, for example as described below in the Examples. However they mayalso be made by synthetic means using techniques well known to skilledpersons such as solid phase synthesis. The proteins may also be producedas fusion proteins, for example to aid in extraction and purification.Examples of fusion protein partners include glutathione-S-transferase(GST), 6×His, GAL4 (DNA binding and/or transcriptional activationdomains) and β-galactosidase. It may also be convenient to include aproteolytic cleavage site between the fusion protein partner and theprotein sequence of interest to allow removal of fusion proteinsequences. Preferably the fusion protein will not hinder the function ofthe protein of interest sequence. Proteins may also be obtained bypurification of cell extracts from animal cells.

[0119] The rSPD(n/CRD) polypeptides, variants, homologues, fragments andderivatives disclosed here may be in a substantially isolated form. Itwill be understood that such polypeptides may be mixed with carriers ordiluents which will not interfere with the intended purpose of theprotein and still be regarded as substantially isolated. A rSPD(n/CRD)variant, homologue, fragment or derivative may also be in asubstantially purified form, in which case it will generally comprisethe protein in a preparation in which more than 90%, e.g. 95%, 98% or99% of the protein in the preparation is a protein.

[0120] The rSPD(n/CRD) polypeptides, variants, homologues, fragments andderivatives disclosed here may be labelled with a revealing label. Therevealing label may be any suitable label which allows the polypeptide,etc. to be detected. Suitable labels include radioisotopes, e.g. ¹²⁵I,enzymes, antibodies, polynucleotides and linkers such as biotin.Labelled polypeptides may be used in diagnostic procedures such asimmunoassays to determine the amount of a polypeptide in a sample.Polypeptides or labelled polypeptides may also be used in serological orcell-mediated immune assays for the detection of immune reactivity tosaid polypeptides in animals and humans using standard protocols.

[0121] A rSPD(n/CRD) polypeptides, variants, homologues, fragments andderivatives disclosed here, optionally labelled, my also be fixed to asolid phase, for example the surface of an immunoassay well or dipstick.Such labelled and/or immobilised polypeptides may be packaged into kitsin a suitable container along with suitable reagents, controls,instructions and the like. Such polypeptides and kits may be used inmethods of detection of antibodies to the polypeptides or their allelicor species variants by immunoassay.

[0122] Immunoassay methods are well known in the art and will generallycomprise: (a) providing a polypeptide comprising an epitope bindable byan antibody against said protein; (b) incubating a biological samplewith said polypeptide under conditions which allow for the formation ofan antibody-antigen complex; and (c) determining whetherantibody-antigen complex comprising said polypeptide is formed.

[0123] The rSPD(n/CRD) polypeptides, variants, homologues, fragments andderivatives disclosed here may be used in in vitro or in vivo cellculture systems to study the role of their corresponding genes andhomologues thereof in cell function, including their function indisease. For example, truncated or modified polypeptides may beintroduced into a cell to disrupt the normal functions which occur inthe cell. The polypeptides may be introduced into the cell by in situexpression of the polypeptide from a recombinant expression vector (seebelow). The expression vector optionally carries an inducible promoterto control the expression of the polypeptide.

[0124] The use of appropriate host cells, such as insect cells ormammalian cells, is expected to provide for such post-translationalmodifications (e.g. myristolation, glycosylation, truncation, lapidationand tyrosine, serine or threonine phosphorylation) as may be needed toconfer optimal biological activity on recombinant expression products.Such cell culture systems in which the rSPD(n/CRD) polypeptides,variants, homologues, fragments and derivatives disclosed here areexpressed may be used in assay systems to identify candidate substanceswhich interfere with or enhance the functions of the polypeptides in thecell.

[0125] Recombinant Surfactant Protein D rSPD(n/CRD) Nucleic Acids

[0126] We provide for a nucleic acid encoding a rSPD(n/CRD) polypeptide,which we refer to as a “rSPD(n/CRD) nucleic acid”. We also providenucleic acids encoding variants, homologues, derivatives and fragmentsof rSPD(n/CRD), as well as fragments, homologues, derivatives andvariants of rSPD(n/CRD) nucleic acids.

[0127] Preferably, the rSPD(n/CRD) nucleic acid is derived from anatural SP-D sequence, for example, the human SP-D sequence shown in SEQID NO: 3. More preferably, the rSPD(n/CRD) nucleic acid lacks sequenceencoding the N-terminal domain and/or the collagen domain, preferablyboth. In a preferred embodiment, the rSPD(n/CRD) nucleic acid is arecombinant fragment of SP-D, preferably human SP-D depicted in SEQ IDNO: 3, which lacks substantially lacks residues 1-594, or any fragment,homologue, variant or derivative thereof. In a further preferredembodiment, the rSPD(n/CRD) nucleic acid is a recombinant fragment ofSP-D, preferably human SP-D sequence shown in SEQ ID NO: 3, comprisingsubstantially residues 595-1128. Fragments, homologues, variants andderivatives of each of the above sequences are also included.

[0128] In a preferred embodiment, a triplet encoding the proline residuecorresponding to position 200 of the human SP-D sequence (SEQ ID NO: 4)is replaced by a codon encoding another residue. Preferably, the prolineresidue is replaced with a uncharged polar residue, for example, acysteine, serine, threonine or methionine residue. In a highly preferredembodiment, the proline residue is replaced with a serine residue. Thus,preferably the rSPD(n/CRD) nucleic acid may comprise a codon encodingserine at position 598 to 560 of the human SP-D sequence shown in SEQ IDNO: 3. Such a replacement codon may therefore include AGC, AGT, TCA,TCC, TCG and TCT. Most preferably the replacement codon comprises AGC.

[0129] Preferably, such a rSPD(n/CRD) nucleic acid encodes therSPD(n/CRD) polypeptide having the sequence shown in SEQ ID NO: 1. ArSPD(n/CRD) nucleic acid preferably comprises, more preferably consistsof, a sequence as set out in SEQ ID NO: 2.

[0130] As used here in this document, the terms “polynucleotide”,“nucleotide”, and nucleic acid are intended to be synonymous with eachother. “Polynucleotide” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedBNA or DNA. “Polynucleotides” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term polynucleotide also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications has been made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short polynucleotides,often referred to as oligonucleotides.

[0131] It will be understood by a skilled person that numerous differentpolynucleotides and nucleic acids can encode the same polypeptide as aresult of the degeneracy of the genetic code. In addition, it is to beunderstood that skilled persons may, using routine techniques, makenucleotide substitutions that do not affect the polypeptide sequenceencoded by the polynucleotides described here to reflect the codon usageof any particular host organism in which the polypeptides are to beexpressed.

[0132] rSPD(n/CRD) nucleic acids, variants, fragments, derivatives andhomologues may comprise DNA or RNA. They may be single-stranded ordouble-stranded. They may also be polynucleotides which include withinthem synthetic or modified nucleotides. A number of different types ofmodification to oligonucleotides are known in the art. These includemethylphosphonate and phosphorothioate backbones, addition of acridineor polylysine chains at the 3′ and/or 5′ ends of the molecule. For thepurposes of this document, it is to be understood that thepolynucleotides may be modified by any method available in the art. Suchmodifications may be carried out in order to enhance the in vivoactivity or life span of polynucleotides of interest.

[0133] The terms “variant”, “homologue” or “derivative” in relation to anucleotide sequence include any substitution of, variation of,modification of, replacement of, deletion of or addition of one (ormore) nucleic acid from or to the sequence. Preferably said variant,homologues or derivatives code for a polypeptide having biologicalactivity.

[0134] As indicated above, with respect to sequence homology, preferablythere is at least 50 or 75%, more preferably at least 85%, morepreferably at least 90% homology to the sequences shown in the sequencelisting herein. More preferably there is at least 95%, more preferablyat least 98%, homology. Nucleotide homology comparisons may be conductedas described above. A preferred sequence comparison program is the GCGWisconsin Bestfit program described above. The default scoring matrixhas a match value of 10 for each identical nucleotide and −9 for eachmismatch. The default gap creation penalty is −50 and the default gapextension penalty is −3 for each nucleotide.

[0135] We further describe nucleotide sequences that are capable ofhybridising selectively to the sequences presented herein, or anyvariant, fragment or derivative thereof, or to the complement of any ofthe above. Nucleotide sequences are preferably at least 15 nucleotidesin length, more preferably at least 20, 30, 40 or 50 nucleotides inlength.

[0136] The term “hybridization” as used herein shall include “theprocess by which a strand of nucleic acid joins with a complementarystrand through base pairing” as well as the process of amplification ascarried out in polymerase chain reaction technologies.

[0137] Polynucleotides capable of selectively hybridising to thenucleotide sequences presented herein, or to their complement, will begenerally at least 70%, preferably at least 80 or 90% and morepreferably at least 95% or 98% homologous to the correspondingnucleotide sequences presented herein over a region of at least 20,preferably at least 25 or 30, for instance at least 40, 60 or 100 ormore contiguous nucleotides.

[0138] The term “selectively hybridizable” means that the polynucleotideused as a probe is used under conditions where a target polynucleotideis found to hybridize to the probe at a level significantly abovebackground. The background hybridization may occur because of otherpolynucleotides present, for example, in the cDNA or genomic DNA librarybeing screening. In this event, background implies a level of signalgenerated by interaction between the probe and a non-specific DNA memberof the library which is less than 10 fold, preferably less than 100 foldas intense as the specific interaction observed with the target DNA. Theintensity of interaction may be measured, for example, by radiolabellingthe probe, e.g. with ³²P.

[0139] Hybridization conditions are based on the melting temperature(Tm) of the nucleic acid binding complex, as taught in Berger andKimmel, 1987, Guide to Molecular Cloning Techniques, Methods inEnzymology, Vol 152, Academic Press, San Diego, Calif.), and confer adefined “stringency” as explained below.

[0140] Maximum stringency typically occurs at about Tm-5° C. (5° C.below the Tm of the probe); high stringency at about 5° C. to 10° C.below Tm; intermediate stringency at about 10° C. to 20° C. below Tm;and low stringency at about 20° C. to 25° C. below Tm. As will beunderstood by those of skill in the art, a maximum stringencyhybridization can be used to identify or detect identical polynucleotidesequences while an intermediate (or low) stringency hybridization can beused to identify or detect similar or related polynucleotide sequences.

[0141] In a preferred aspect, we provide nucleotide sequences that canhybridise to the rSPD(n/CRD) nucleic acids, fragments, variants,homologues or derivatives under stringent conditions (e.g. 65° C. and0.1×SSC {1×SSC=0.15 M NaCl, 0.015 M Na₃ Citrate pH 7.0).

[0142] Where the polynucleotide is double-stranded, both strands of theduplex, either individually or in combination, are encompassed by themethods and compositions described here. Where the polynucleotide issingle-stranded, it is to be understood that the complementary sequenceof that polynucleotide is also included.

[0143] Polynucleotides which are not 100% homologous to the sequences ofthe present invention but which are also included can be obtained in anumber of ways. Other variants of the sequences may be obtained forexample by probing DNA libraries made from a range of individuals, forexample individuals from different populations. For example, SPDhomologues may be identified from other individuals, or other species.Further recombinant SP-D nucleic acids and polypeptides may be producedby identifying corresponding positions in the homologues, andsynthesising or producing the molecule as described elsewhere in thisdocument. Furthermore, the collagen region, neck region and carbohydratebinding domain in such homologues may be identified, for example, bysequence gazing or computer assisted comparisons, and selected forcombination into or production of a recombinant SP-D which is non-human,but which has one or more biological activities of rSPD(n/CRD).

[0144] In addition, other viral/bacterial, or cellular homologues ofSP-D particularly cellular homologues found in mammalian cells (e.g.rat, mouse, bovine and primate cells), may be obtained and suchhomologues and fragments thereof in general will be capable ofselectively hybridising to human SP-D. Such homologues may be used todesign non-human rSPD(n/CRD) nucleic acids, fragments, variants andhomologues. Mutagenesis may be carried out by means known in the art toproduce further variety.

[0145] Sequences of SP-D homologues may be obtained by probing cDNAlibraries made from or genomic DNA libraries from other animal species,and probing such libraries with probes comprising all or part of any ofthe rSPD(n/CRD) nucleic acids, fragments, variants and homologues, orother fragments of SP-D under conditions of medium to high stringency.

[0146] Similar considerations apply to obtaining species homologues andallelic variants of the polypeptide or nucleotide sequences disclosedhere.

[0147] Variants and strain/species homologues may also be obtained usingdegenerate PCR which will use primers designed to target sequenceswithin the variants and homologues encoding conserved amino acidsequences within the sequences of the rSPD(n/CRD) nucleic acids.Conserved sequences can be predicted, for example, by aligning the aminoacid sequences from several variants/homologues. Sequence alignments canbe performed using computer software known in the art. For example theGCG Wisconsin PileUp program is widely used.

[0148] The primers used in degenerate PCR will contain one or moredegenerate positions and will be used at stringency conditions lowerthan those used for cloning sequences with single sequence primersagainst known sequences. It will be appreciated by the skilled personthat overall nucleotide homology between sequences from distantlyrelated organisms is likely to be very low and thus in these situationsdegenerate PCR may be the method of choice rather than screeninglibraries with labelled fragments the rSPD(n/CRD) sequences.

[0149] In addition, homologous sequences may be identified by searchingnucleotide and/or protein databases using search algorithms such as theBLAST suite of programs.

[0150] Alternatively, such polynucleotides may be obtained by sitedirected mutagenesis of characterised sequences, for example,rSPD(n/CRD) nucleic acids, or variants, homologues, derivatives orfragments thereof. This may be useful where for example silent codonchanges are required to sequences to optimise codon preferences for aparticular host cell in which the polynucleotide sequences are beingexpressed. Other sequence changes may be desired in order to introducerestriction enzyme recognition sites, or to alter the property orfunction of the polypeptides encoded by the polynucleotides.

[0151] The polynucleotides described here may be used to produce aprimer, e.g. a PCR primer, a primer for an alternative amplificationreaction, a probe e.g. labelled with a revealing label by conventionalmeans using radioactive or non-radioactive labels, or thepolynucleotides may be cloned into vectors. Such primers, probes andother fragments will be at least 8, 9, 10, or 15, preferably at least20, for example at least 25, 30 or 40 nucleotides in length, and arealso encompassed by the term “polynucleotides” as used herein.

[0152] Polynucleotides such as a DNA polynucleotides and probes may beproduced recombinantly, synthetically, or by any means available tothose of skill in the art. They may also be cloned by standardtechniques.

[0153] In general, primers will be produced by synthetic means,involving a step wise manufacture of the desired nucleic acid sequenceone nucleotide at a time. Techniques for accomplishing this usingautomated techniques are readily available in the art.

[0154] Longer polynucleotides will generally be produced usingrecombinant means, for example using a PCR (polymerase chain reaction)cloning techniques. This will involve making a pair of primers (e.g. ofabout 15 to 30 nucleotides) flanking a region of the lipid targetingsequence which it is desired to clone, bringing the primers into contactwith mRNA or cDNA obtained from an animal or human cell, performing apolymerase chain reaction under conditions which bring aboutamplification of the desired region, isolating the amplified fragment(e.g. by purifying the reaction mixture on an agarose gel) andrecovering the amplified DNA. The primers may be designed to containsuitable restriction enzyme recognition sites so that the amplified DNAcan be cloned into a suitable cloning vector.

[0155] Polynucleotides or primers may carry a revealing label. Suitablelabels include radioisotopes such as ³²P or ³⁵S, enzyme labels, or otherprotein labels such as biotin. Such labels may be added topolynucleotides or primers and may be detected using by techniques knownper se. Polynucleotides or primers or fragments thereof labelled orunlabeled may be used by a person skilled in the art in nucleicacid-based tests for detecting or sequencing polynucleotides in thehuman or animal body.

[0156] Such tests for detecting generally comprise bringing a biologicalsample containing DNA or RNA into contact with a probe comprising apolynucleotide or primer under hybridising conditions and detecting anyduplex formed between the probe and nucleic acid in the sample. Suchdetection maybe achieved using techniques such as PCR or by immobilisingthe probe on a solid support, removing nucleic acid in the sample whichis not hybridised to the probe, and then detecting nucleic acid whichhas hybridised to the probe. Alternatively, the sample nucleic acid maybe immobilised on a solid support, and the amount of probe bound to sucha support can be detected. Suitable assay methods of this and otherformats can be found in for example WO89/03891 and WO90/13667.

[0157] Tests for sequencing nucleotides, for example, the rSPD(n/CRD)nucleic acids, involve bringing a biological sample containing targetDNA or RNA into contact with a probe comprising a polynucleotide orprimer under hybridising conditions and determining the sequence by, forexample the Sanger dideoxy chain termination method (see Sambrook etal.).

[0158] Such a method generally comprises elongating, in the presence ofsuitable reagents, the primer by synthesis of a strand complementary tothe target DNA or RNA and selectively terminating the elongationreaction at one or more of an A, C, G or T/U residue; allowing strandelongation and termination reaction to occur; separating out accordingto size the elongated products to determine the sequence of thenucleotides at which selective termination has occurred. Suitablereagents include a DNA polymerase enzyme, the deoxynucleotides dATP,dCTP, dGTP and dTTP, a buffer and ATP. Dideoxynucleotides are used forselective termination.

[0159] Nucleic Acid Vectors

[0160] Polynucleotides, for example those described here, can beincorporated into a recombinant replicable vector. The vector may beused to replicate the nucleic acid in a compatible host cell. Thus in afurther embodiment, we provide a method of making polynucleotides byintroducing a polynucleotide into a replicable vector, introducing thevector into a compatible host cell, and growing the host cell underconditions which bring about replication of the vector. The vector maybe recovered from the host cell. Suitable host cells include bacteriasuch as E. coli, yeast, mammalian cell lines and other eukaryotic celllines, for example insect Sf9 cells.

[0161] Preferably, a polynucleotide in a vector is operably linked to acontrol sequence that is capable of providing for the expression of thecoding sequence by the host cell, i.e. the vector is an expressionvector. The term “operably linked” means that the components describedare in a relationship permitting them to function in their intendedmanner. A regulatory sequence “operably linked” to a coding sequence isligated in such a way that expression of the coding sequence is achievedunder condition compatible with the control sequences.

[0162] The control sequences may be modified, for example by theaddition of further transcriptional regulatory elements to make thelevel of transcription directed by the control sequences more responsiveto transcriptional modulators.

[0163] Vectors may be transformed or transfected into a suitable hostcell as described below to provide for expression of a protein. Thisprocess may comprise culturing a host cell transformed with anexpression vector as described above under conditions to provide forexpression by the vector of a coding sequence encoding the protein, andoptionally recovering the expressed protein. Vectors will be chosen thatare compatible with the host cell used.

[0164] The vectors may be for example, plasmid or virus vectors providedwith an origin of replication, optionally a promoter for the expressionof the said polynucleotide and optionally a regulator of the promoter.The vectors may contain one or more selectable marker genes, for examplean ampicillin resistance gene in the case of a bacterial plasmid or aneomycin resistance gene for a mammalian vector. Vectors may be used,for example, to transfect or transform a host cell.

[0165] Control sequences operably linked to sequences encoding thepolypeptide include promoters/enhancers and other expression regulationsignals. These control sequences may be selected to be compatible withthe host cell for which the expression vector is designed to be used in.The term promoter is well-known in the art and encompasses nucleic acidregions ranging in size and complexity from minimal promoters topromoters including upstream elements and enhancers.

[0166] The promoter is typically selected from promoters which arefunctional in mammalian cells, although prokaryotic promoters andpromoters functional in other eukaryotic cells, such as insect cells,may be used. The promoter is typically derived from promoter sequencesof viral or eukaryotic genes. For example, it may be a promoter derivedfrom the genome of a cell in which expression is to occur. With respectto eukaryotic promoters, they may be promoters that function in aubiquitous manner (such as promoters of α-actin, β-actin, tubulin) or,alternatively, a tissue-specific manner (such as promoters of the genesfor pyruvate kinase). They may also be promoters that respond tospecific stimuli, for example promoters that bind steroid hormonereceptors. Viral promoters may also be used, for example the Moloneymurine leukaemia virus long terminal repeat (MMLV LTR) promoter, therous sarcoma virus (RSV) LTR promoter or the human cytomegalovirus (CMV)IE promoter.

[0167] It may also be advantageous for the promoters to be inducible sothat the levels of expression of the heterologous gene can be regulatedduring the life-time of the cell. Inducible means that the levels ofexpression obtained using the promoter can be regulated.

[0168] In addition, any of these promoters may be modified by theaddition of further regulatory sequences, for example enhancersequences. Chimeric promoters may also be used comprising sequenceelements from two or more different promoters described above.

[0169] Polynucleotides may also be inserted into the vectors describedabove in an antisense orientation to provide for the production ofantisense RNA. Antisense RNA or other antisense polynucleotides may alsobe produced by synthetic means. Such antisense polynucleotides may beused in a method of controlling the levels of RNAs transcribed fromgenes comprising any one of the polynucleotides described here.

[0170] Host Cells

[0171] Vectors and polynucleotides comprising or encoding rSPD(n/CRD)nucleic acids, fragments, homologues, variants or derivatives thereofmay be introduced into host cells for the purpose of replicating thevectors/polynucleotides and/or expressing the polypeptides encoded bythe polynucleotides. Although the polypeptides may be produced usingprokaryotic cells as host cells, it is preferred to use eukaryoticcells, for example yeast, insect or mammalian cells, in particularmammalian cells.

[0172] Vectors/polynucleotides may be introduced into suitable hostcells using a variety of techniques known in the art, such astransfection, transformation and electroporation. Wherevectors/polynucleotides are to be administered to animals, severaltechniques are known in the art, for example infection with recombinantviral vectors such as retroviruses, herpes simplex viruses andadenoviruses, direct injection of nucleic acids and biolistictransformation.

[0173] Protein Expression and Purification

[0174] Host cells comprising polynucleotides may be used to expresspolypeptides, such as rSPD(n/CRD) polypeptides, fragments, homologues,variants or derivatives thereof. Host cells may be cultured undersuitable conditions which allow expression of the proteins. Expressionof the polypeptides may be constitutive such that they are continuallyproduced, or inducible, requiring a stimulus to initiate expression. Inthe case of inducible expression, protein production can be initiatedwhen required by, for example, addition of an inducer substance to theculture medium, for example dexamethasone or IPTG.

[0175] Polypeptides can be extracted from host cells by a variety oftechniques known in the art, including enzymatic, chemical and/orosmotic lysis and physical disruption.

[0176] Polypeptides may also be produced recombinantly in an in vitrocell-free system, such as the TnT™ (Promega) rabbit reticulocyte system.

[0177] Antibodies

[0178] The invention also provides monoclonal or polyclonal antibodiesto polypeptides or fragments thereof. Thus, the present inventionfurther provides a process for the production of monoclonal orpolyclonal antibodies to an rSPD(n/CRD) polypeptide, fragment,homologue, variant or derivative thereof.

[0179] If polyclonal antibodies are desired, a selected mammal (e.g.,mouse, rabbit, goat, horse, etc.) is immunised with an immunogenicpolypeptide bearing an epitope(s) from a polypeptide. Serum from theimmunised animal is collected and treated according to known procedures.If serum containing polyclonal antibodies to an epitope from apolypeptide contains antibodies to other antigens, the polyclonalantibodies can be purified by immunoaffinity chromatography. Techniquesfor producing and processing polyclonal antisera are known in the art.In order that such antibodies may be made, the invention also providespolypeptides or fragments thereof haptenised to another polypeptide foruse as immunogens in animals or humans.

[0180] Monoclonal antibodies directed against epitopes in thepolypeptides can also be readily produced by one skilled in the art. Thegeneral methodology for making monoclonal antibodies by hybridomas iswell known. Immortal antibody-producing cell lines can be created bycell fusion, and also by other techniques such as direct transformationof B lymphocytes with oncogenic DNA, or transfection with Epstein-Barrvirus. Panels of monoclonal antibodies produced against epitopes in thepolypeptides can be screened for various properties; i.e., for isotypeand epitope affinity.

[0181] An alternative technique involves screening phage displaylibraries where, for example the phage express scFv fragments on thesurface of their coat with a large variety of complementaritydetermining regions (CDRs). This technique is well known in the art.

[0182] Antibodies, both monoclonal and polyclonal, which are directedagainst epitopes from polypeptides are particularly useful in diagnosis,and those which are neutralising are useful in passive immunotherapy.Monoclonal antibodies, in particular, may be used to raise anti-idiotypeantibodies. Anti-idiotype antibodies are immunoglobulins which carry an“internal image” of the antigen of the agent against which protection isdesired.

[0183] Techniques for raising anti-idiotype antibodies are known in theart. These anti-idiotype antibodies may also be useful in therapy.

[0184] For the purposes of this invention, the term “antibody”, unlessspecified to the contrary, includes fragments of whole antibodies whichretain their binding activity for a target antigen. Such fragmentsinclude Fv, F(ab′) and F(ab′)₂ fragments, as well as single chainantibodies (scFv). Furthermore, the antibodies and fragments thereof maybe humanised antibodies, for example as described in EP-A-239400.

[0185] Antibodies may be used in method of detecting polypeptidespresent in biological samples by a method which comprises: (a) providingan antibody; (b) incubating a biological sample with said antibody underconditions which allow for the formation of an antibody-antigen complex;and (c) determining whether antibody-antigen complex comprising saidantibody is formed.

[0186] Suitable samples include extracts tissues such as brain, breast,ovary, lung, colon, pancreas, testes, liver, muscle and bone tissues orfrom neoplastic growths derived from such tissues.

[0187] Antibodies may be bound to a solid support and/or packaged intokits in a suitable container along with suitable reagents, controls,instructions and the like.

[0188] Assays

[0189] We disclose assays that are suitable for identifying substanceswhich bind to rSPD(n/CRD) polypeptides, or fragments, homologues,variants or derivatives thereof.

[0190] In general, such binding assays involve exposing a rSPD(n/CRD)polypeptide, nucleic acid, or a fragment, homologue, variant orderivative thereof to a candidate molecule and detecting an interactionor binding between the rSPD(n/CRD) polypeptide, nucleic acid, or afragment, homologue, variant or derivative thereof and the candidatemolecule. The binding assay may be conducted in vitro, or in vivo.

[0191] We disclose assays for identifying substances which are capableof potentiating the activities of rSPD(n/CRD) polypeptide. Suchcompounds may be employed as agonists of rSPD(n/CRD) polypeptide, andmay for example be co-administered to an individual to enhance anydesired effect.

[0192] In general, an assay to identify such substances or compoundsinvolves providing a cell or organism, exposing the cell or organism toa rSPD(n/CRD) polypeptide, nucleic acid, or a fragment, homologue,variant or derivative thereof, exposing the cell to a candidatemolecule, and detecting an effect associated with rSPD(n/CRD). AnyrSPD(n/CRD) polypeptide mediated effect, as disclosed in this document,particularly the Examples, may be detected.

[0193] In particular, the rSPD(n/CRD) polypeptide mediated effect ispreferably chosen from the group consisting of: reduction of peripheralblood eosinophilia, reduction of serum IgB levels, reduction of serumIgG1 levels, reduction in airway hyperresponsiveness, reduction inalveolar macrophage number, reduction of phospholipid levels in lavage,down-regulation of Eotaxin expression, reduction in MCP-1 expression,down-regulation of MIP-1α expression, and down-regulation of MIP-2expression.

[0194] In order to identify agonists, an additive or preferablysynergistic effect is detected. Thus, while rSPD(n/CRD) polypeptide onits own is, for example, capable of reducing a level or number, ordown-regulation of expression of a molecule, the assays identifymolecules which further reduce the level, number or furtherdown-regulate the expression of a molecule. Thus, preferably, thecandidate molecule in conjunction with the rSPD(n/CRD) polypeptide,nucleic acid, or a fragment, homologue, variant or derivative thereof,down-regulates the expression of, or reduces the level or number, bymore than 10%, more than 20%, more than 30%, more than 40%, more than50%, more than 60%, more than 70%, more than 80%, more than 90%, or morecompared to an rSPD(n/CRD) polypeptide on its own. Thus, for example, acandidate molecule suitable for use as an agonist is one which iscapable of enhancing by 10% more the reduction of alveolar macrophagenumber achieved by rSPD(n/CRD) polypeptide on its own.

[0195] Conversely, assays to identify antagonists involve the detectionof a reduction in rSPD(n/CRD) polypeptide mediated effect. Preferably,the down-regulation of expression or reduction in number or levelachieved by rSPD(n/CRD) polypeptide is reduced in the presence of asuitable candidate molecule. Preferably, the reduction is at least 10%,preferably at least 20%, preferably at least 30%, preferably at least40%, preferably at least 50%, preferably at least 60%, preferably atleast 70%, preferably at least 80%, preferably at least 90%, or morecompared to an rSPD(n/CRD) polypeptide on its own. Thus, for example, acandidate molecule suitable for use as an antagonist is one which iscapable of reducing by 10% more the reduction of alveolar macrophagenumber achieved by rSPD(n/CRD) polypeptide on its own.

[0196] As an illustration, if N1 is the number of alveolar macrophagesin an untreated organism, and N2 the number in an organism exposed torSPD(n/CRD) polypeptide, nucleic acid, or a fragment, homologue, variantor derivative thereof, the alveolar macrophage number is decreased byR=(N1−N2)/N1×100%. Agonists increase R, by a factor x, where x isgreater than 1 (e.g., x=1, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 10, 20,50, 100 etc); while antagonists decrease R, by a factor x, where x isless than 1 (e.g., x=0.9, 0.9, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 etc).

[0197] For example, an organism may be exposed to a rSPD(n/CRD)polypeptide, nucleic acid, or a fragment, homologue, variant orderivative thereof and a candidate molecule, and the peripheraleosinophil level, or serum IgE or serum IgG1, or any combination,detected. Detection of airway hyperresponsiveness, or alvelar macrophagenumbers, of an organism exposed to an rSPD(n/CRD) polypeptide, nucleicacid, or a fragment, homologue, variant or derivative thereof, togetherwith a candidate molecule, may also be detected.

[0198] Preferred candidate molecules are those which provide an additiveor synergistic effect in combination with rSPD(n/CRD).

[0199] Also disclosed are assays to identify antagonists of rSPD(n/CRD)polypeptide. Such assays involve detecting a reduced effect on exposureof a cell or organism to an rSPD(n/CRD) polypeptide, nucleic acid, or afragment, homologue, variant or derivative thereof in conjunction with acandidate molecule.

[0200] In a preferred embodiment, the assays are conducted on wholeorganisms rather than cells. Preferably, the organism is one whichsuffers from a disease as disclosed in this document, or exhibits one ormore symptoms of such a disease. In a highly preferred embodiment, theorganism is one which does not express surfactant protein D. Preferably,such an organism comprises a disrupted or deleted SP-D gene. Morepreferably, the organism is a mouse (Mus musculus), preferably a SP-Dknock-out mouse, made as described in Botas, C. et al., 1998, Alteredsurfactant homeostasis and alveolar type II cell morphology in micelacking surfactant protein D. Proc. Natl. Acad. Sci. USA 95(20):11869-74.

[0201] Candidate Molecules

[0202] Suitable candidate molecules for use in the above assays includepeptides, especially of from about 5 to 30 or 10 to 25 amino acids insize. Peptides from panels of peptides comprising random sequences orsequences which have been varied consistently to provide a maximallydiverse panel of peptides may be used.

[0203] Suitable candidate molecules also include antibody products (forexample, monoclonal and polyclonal antibodies, single chain antibodies,chimeric antibodies and CDR-grafted antibodies). Furthermore,combinatorial libraries, peptide and peptide mimetics, defined chemicalentities, oligonucleotides, and natural product libraries may bescreened for activity. The candidate molecules may be used in an initialscreen in batches of, for example 10 types of molecules per reaction,and the molecules of those batches which show enhancement or reductionof a rSPD(n/CRD) polypeptide mediated effect tested individually.

[0204] Libraries

[0205] Libraries of candidate molecules, such as libraries ofpolypeptides or nucleic acids, may be employed in the methods andcompositions described here. Such libraries are exposed a cell ororganism in the presence of a rSPD(n/CRD) polypeptide, nucleic acid, ora fragment, homologue, variant or derivative thereof, and an rSPD(n/CRD)polypeptide mediated effect detected.

[0206] Selection protocols for isolating desired members of largelibraries are known in the art, as typified by phage display techniques.Such systems, in which diverse peptide sequences are displayed on thesurface of filamentous bacteriophage (Scott and Smith, 1990, supra),have proven useful for creating libraries of antibody fragments (and thenucleotide sequences that encoding them) for the in vitro selection andamplification of specific antibody fragments that bind a target antigen.The nucleotide sequences encoding the V_(H) and V_(L) regions are linkedto gene fragments which encode leader signals that direct them to theperiplasmic space of E. coli and as a result the resultant antibodyfragments are displayed on the surface of the bacteriophage, typicallyas fusions to, bacteriophage coat proteins (e.g., pIII or pVIII).Alternatively, antibody fragments are displayed externally on lambdaphage capsids (phagebodies). An advantage of phage-based display systemsis that, because they are biological systems, selected library memberscan be amplified simply by growing the phage containing the selectedlibrary member in bacterial cells. Furthermore, since the nucleotidesequence that encodes the polypeptide library member is contained on aphage or phagemid vector, sequencing, expression and subsequent geneticmanipulation is relatively straightforward.

[0207] Methods for the construction of bacteriophage antibody displaylibraries and lambda phage expression libraries are well known in theart (McCafferty et al. ,1990, supra; Kang et al., 1991, Proc. Natl.Acad. Sci. USA 88:4363; Clackson et al., 1991, Nature 352:624; Lowman etal., 1991, Biochemistry 30:10832; Burton et al, 1991, Proc. Natl. Acad.Sci. USA 88:10134; Hoogenboom et al., 1991, Nucleic Acids Res. 19:4133;Chang et al., 1991, J. Immunol. 147:3610; Breitling et al. ,1991, Gene104:147; Marks et al., 1991, supra; Barbas et al., 1992, supra; Hawkinsand Winter, 1992, J. Immunol. 22:867; Marks et al., 1992, J. Biol. Chem.267:16007; Lemer et al., 1992, Science 258:1313, incorporated herein byreference). Such techniques may be modified if necessary for theexpression generally of polypeptide libraries.

[0208] One particularly advantageous approach has been the use of scFvphage-libraries (Bird, R. E. et al., 1988, Science 242:423-6, Huston etal., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Chaudhary et al.,1990, Proc. Natl. Acad. Sci. USA 87:1066-1070; McCafferty et al., 1990,supra; Clackson et al., 1991, supra; Marks et al., 1991, supra; Chiswellet al, 1992, Trends Biotech. 10:80; Marks et al., 1992, supra). Variousembodiments of scFv libraries displayed on bacteriophage coat proteinshave been described. Refinements of phage display approaches are alsoknown, for example as described in WO96/06213 and WO92/01047 (MedicalResearch Council et al.) and WO97/08320 (Morphosys, supra), which areincorporated herein by reference.

[0209] Alternative library selection technologies include bacteriophagelambda expression systems, which may be screened directly asbacteriophage plaques or as colonies of lysogens, both as previouslydescribed (Huse et al., 1989, Science 246:1275; Caton and Koprowski,1990, Proc. Natl. Acad. Sci. USA 87; Mullinax et al., 1990, Proc. Natl.Acad. Sci. USA 87:8095; Persson et al., 1991, Proc. Natl. Acad. Sci. USA88:2432) and are of use in the invention. These expression systems maybe used to screen a large number of different members of a library, inthe order of about 10⁶ or even more. Other screening systems rely, forexample, on direct chemical synthesis of library members. One earlymethod involves the synthesis of peptides on a set of pins or rods, suchas described in WO84/03564. A similar method involving peptide synthesison beads, which forms a peptide library in which each bead is anindividual library member, is described in U.S. Pat. No. 4,631,211 and arelated method is described in WO92/00091. A significant improvement ofthe bead-based methods involves tagging each bead with a uniqueidentifier tag, such as an oligonucleotide, so as to facilitateidentification of the amino acid sequence of each library member. Theseimproved bead-based methods are described in WO93/06121.

[0210] Another chemical synthesis method involves the synthesis ofarrays of peptides (or peptidomimetics) on a surface in a manner thatplaces each distinct library member (e.g., unique peptide sequence) at adiscrete, predefined location in the array. The identity of each librarymember is determined by its spatial location in the array. The locationsin the array where binding interactions between a predetermined molecule(e.g., a receptor) and reactive library members occur is determined,thereby identifying the sequences of the reactive library members on thebasis of spatial location. These methods are described in U.S. Pat. No.5,143,854; WO90/15070 and WO92/10092; Fodor et al., 1991, Science251:767; Dower and Fodor, 1991, Ann. Rep. Med. Chem. 26:271.

[0211] Other systems for generating libraries of polypeptides ornucleotides involve the use of cell-free enzymatic machinery for the invitro synthesis of the library members. In one method, RNA molecules areselected by alternate rounds of selection against a target ligand andPCR amplification (Tuerk and Gold, 1990, Science 249:505; Ellington andSzostak, 1990, Nature 346:818). A similar technique may be used toidentify DNA sequences which bind a predetermined human transcriptionfactor (Thiesen and Bach, 1990, Nucleic Acids Res. 18:3203; Beaudry andJoyce, 1992, Science 257:635; WO92/05258 and WO92/14843). In a similarway, in vitro translation can be used to synthesise polypeptides as amethod for generating large libraries. These methods which generallycomprise stabilised polysome complexes, are described further inWO88/08453, WO90/05785, WO90/07003, WO91/02076, WO91/05058, andWO92/02536. Alternative display systems which are not phage-based, suchas those disclosed in WO95/22625 and WO95/11922 (Affymax) use thepolysomes to display polypeptides for selection. These and all theforegoing documents also are incorporated herein by reference.

[0212] Combinatorial Libraries

[0213] Libraries, in particular, libraries of candidate molecules, maysuitably be in the form of combinatorial libraries (also known ascombinatorial chemical libraries).

[0214] A “combinatorial library”, as the term is used in this document,is a collection of multiple species of chemical compounds that consistof randomly selected subunits. Combinatorial libraries maybe screenedfor molecules which are capable of potentiating, enhancing, reducing orminimising the a rSPD(n/CRD) polypeptide mediated effect when exposed toa cell or organism.

[0215] Various combinatorial libraries of chemical compounds arecurrently available, including libraries active against proteolytic andnon-proteolytic enzymes, libraries of agonists and antagonists ofG-protein coupled receptors (GPCRs), libraries active against non-GPCRtargets (e.g., integrins, ion channels, domain interactions, nuclearreceptors, and transcription factors) and libraries of whole-celloncology and anti-infective targets, among others. A comprehensivereview of combinatorial libraries, in particular their construction anduses is provided in Dolle and Nelson, 1999, Journal of CombinatorialChemistry Vol 1 No 4:235-282. Reference is also made to Combinatorialpeptide library protocols (edited by Shmuel Cabilly, Totowa, N.J. HumanaPress, c1998, Methods in Molecular Biology, v. 87).

[0216] Further references describing chemical combinatorial libraries,their production and use include those available from the URLhttp://www.netsci.org/Science/Combichem/, including The ChemicalGeneration of Molecular Diversity, Pavia, Michael R., SphinxPharmaceuticals, A Division of Eli Lilly (Published July 1995);Combinatorial Chemistry: A Strategy for the Future—MDL InformationSystems discusses the role its Project Library plays in managingdiversity libraries (Published July 1995); Solid Support CombinatorialChemistry in Lead Discovery and SAR Optimization, Mjalli, Andan M. M.and Barry E. Toyonaga, Ontogen Corporation (Published July 1995);Non-Peptidic Bradykinin Receptor Antagonists From a StructurallyDirected Non-Peptide Library, Chakravarty, Sarvajit, Babu J. Mavunkel,Robin Andy, and Donald J. Kyle*, Scios Nova Inc. (Published July 1995);Combinatorial Chemistry Library Design using Pharmacophore Diversity,Davies, Keith and Clive Briant, Chemical Design Ltd. (Published July1995); A Database System for Combinatorial Synthesis Experiments, James,Craig and David Weininger, Daylight Chemical Information Systems, Inc.(Published July 1995); An Information Management Architecture forCombinatorial Chemistry, Davies, Keith and Catherine White, ChemicalDesign Ltd. (Published July 1995); Novel Software Tools for AddressingChemical Diversity, Pearlman, R. S., Laboratory for Molecular Graphicsand Theoretical Modeling, College of Pharmacy, University of Texas(Published June/July 1996); Opportunities for Computational ChemistsAfforded by the New Strategies in Drug Discovery: An Opinion, ConnollyMartin, Yvonne, Computer Assisted Molecular Design Project, AbbottLaboratories (Published June/July 1996); Combinatorial Chemistry andMolecular Diversity Course at the University of Louisville: ADescription, Spatola, Arno F., Department of Chemistry, University ofLouisville (Published June/July 1996); Chemically Generated ScreeningLibraries: Present and Future, Pavia, Michael R., SphinxPharmaceuticals, A Division of Eli Lilly (Published June/July 1996);Chemical Strategies For Introducing Carbohydrate Molecular DiversityInto The Drug Discovery Process, Sofia, Michael J., TranscellTechnologies Inc. (Published June/July 1996); Data Management forCombinatorial Chemistry, Zaborowski, Maryjo, Chiron Corporation andSheila H. DeWitt, Parke-Davis Pharmaceutical Research, Division ofWarner-Lambert Company (Published November 1995); and The Impact of HighThroughput Organic Synthesis on R&D in Bio-Based Industries, Devlin,John P. (Published March 1996).

[0217] Techniques in combinatorial chemistry are gaining wide acceptanceamong modem methods for the generation of new pharmaceutical leads(Gallop, M. A. et al., 1994, J. Med. Chem. 37:1233-1251; Gordon, E. M.et al., 1994, J. Med. Chem. 37:1385-1401.). One combinatorial approachin use is based on a strategy involving the synthesis of librariescontaining a different structure on each particle of the solid phasesupport, interaction of the library with a soluble receptor,identification of the “bead” which interacts with the macromoleculartarget, and determination of the structure carried by the identified“bead” (Lam, K. S. et al., 1991, Nature 354:82-84). An alternative tothis approach is the sequential release of defined aliquots of thecompounds from the solid support, with subsequent determination ofactivity in solution, identification of the particle from which theactive compound was released, and elucidation of its structure by directsequencing (Salmon, S. E. et al., 1993, Proc. Natl. Acad. Sci. USA90:11708-11712), or by reading its code (Kerr, J. M. et al., 1993, J.Am. Chem. Soc. 115:2529-2531; Nikolaiev, V. et al., 1993, Pept. Res.6:161-170; Ohlmeyer, M. H. J. et al., 1993, Proc. Natl. Acad. Sci. USA90:10922-10926).

[0218] Soluble random combinatorial libraries maybe synthesized using asimple principle for the generation of equimolar mixtures of peptideswhich was first described by Furka (Furka, A. et al., 1988, XthInternational Symposium on Medicinal Chemistry, Budapest 1988; Furka, A.et al., 1988, 14th International Congress of Biochemistry, Prague 1988;Furka, A. et al., 1991, Int. J. Peptide Protein Res. 37:487-493). Theconstruction of soluble libraries for iterative screening has also beendescribed (Houghten, R. A. et al., 1991, Nature 354:84-86). K. S. Lamdisclosed the novel and unexpectedly powerful technique of usinginsoluble random combinatorial libraries. Lam synthesized randomcombinatorial libraries on solid phase supports, so that each supporthad a test compound of uniform molecular structure, and screened thelibraries without prior removal of the test compounds from the supportby solid phase binding protocols (Lam, K. S. et al., 1991, Nature354:82-84).

[0219] Thus, a library of candidate molecules may be a syntheticcombinatorial library (e.g., a combinatorial chemical library), acellular extract, a bodily fluid (e.g., urine, blood, tears, sweat, orsaliva), or other mixture of synthetic or natural products (e.g., alibrary of small molecules or a fermentation mixture).

[0220] A library of molecules may include, for example, amino acids,oligopeptides, polypeptides, proteins, or fragments of peptides orproteins; nucleic acids (e.g., antisense; DNA; RNA; or peptide nucleicacids, PNA); aptamers; or carbohydrates or polysaccharides. Each memberof the library can be singular or can be a part of a mixture (e.g., acompressed library). The library may contain purified compounds or canbe “dirty” (i.e., containing a significant quantity of impurities).Commercially available libraries (e.g., from Affymetrix, ArQule, NeoseTechnologies, Sarco, Ciddco, Oxford Asymmetry, Maybridge, Aldrich,Panlabs, Pharmacopoeia, Sigma, or Tripose) may also be used with themethods described here.

[0221] In addition to libraries as described above, special librariescalled diversity files can be used to assess the specificity,reliability, or reproducibility of the new methods. Diversity filescontain a large number of compounds (e.g., 1000 or more small molecules)representative of many classes of compounds that could potentiallyresult in nonspecific detection in an assay. Diversity files arecommercially available or can also be assembled from individualcompounds commercially available from the vendors listed above.

[0222] Candidate Substances

[0223] Suitable candidate substances include peptides, especially offrom about 5 to 30 or 10 to 25 amino acids in size, based on thesequence of the polypeptides described in the Examples, or variants ofsuch peptides in which one or more residues have been substituted.Peptides from panels of peptides comprising random sequences orsequences which have been varied consistently to provide a maximallydiverse panel of peptides may be used.

[0224] Suitable candidate substances also include antibody products (forexample, monoclonal and polyclonal antibodies, single chain antibodies,chimeric antibodies and CDR-grafted antibodies) which are specific for apolypeptide. Furthermore, combinatorial libraries, peptide and peptidemimetics, defined chemical entities, oligonucleotides, and naturalproduct libraries may be screened for activity as inhibitors of bindingof a polypeptide to the cell division cycle machinery, for examplemitotic/meiotic apparatus (such as microtubules). The candidatesubstances may be used in an initial screen in batches of, for example10 substances per reaction, and the substances of those batches whichshow inhibition tested individually. Candidate substances which showactivity in in vitro screens such as those described below can then betested in whole cell systems, such as mammalian cells which will beexposed to the inhibitor and tested for inhibition of any of the stagesof the cell cycle.

[0225] Polypeptide Binding Assays

[0226] One type of assay for identifying substances that bind to apolypeptide involves contacting a polypeptide, which is immobilised on asolid support, with a non-immobilised candidate substance determiningwhether and/or to what extent the polypeptide and candidate substancebind to each other. Alternatively, the candidate substance may beimmobilised and the polypeptide non-immobilised. This may be used todetect substances capable of binding to rSPD(n/CRD) polypeptides, orfragments, homologues, variants or derivatives thereof.

[0227] In a preferred assay method, the polypeptide is immobilised onbeads such as agarose beads. Typically this is achieved by expressingthe rSPD(n/CRD) polypeptide, or a fragment, homologue, variant orderivative thereof as a GST-fusion protein in bacteria, yeast or highereukaryotic cell lines and purifying the GST-fusion protein from crudecell extracts using glutathione-agarose beads (Smith and Johnson, 1988).As a control, binding of the candidate substance, which is not aGST-fusion protein, to the immobilised polypeptide is determined in theabsence of the polypeptide. The binding of the candidate substance tothe immobilised polypeptide is then determined. This type of assay isknown in the art as a GST pulldown assay. Again, the candidate substancemay be immobilised and the polypeptide non-immobilised.

[0228] It is also possible to perform this type of assay using differentaffinity purification systems for immobilising one of the components,for example Ni-NTA agarose and histidine-tagged components.

[0229] Binding of the rSPD(n/CRD) polypeptide, or a fragment, homologue,variant or derivative thereof to the candidate substance may bedetermined by a variety of methods well-known in the art. For example,the non-immobilised component may be labeled (with for example, aradioactive label, an epitope tag or an enzyme-antibody conjugate).Alternatively, binding may be determined by immunological detectiontechniques. For example, the reaction mixture can be Western blotted andthe blot probed with an antibody that detects the non-immobilisedcomponent. ELISA techniques may also be used.

[0230] Candidate substances are typically added to a final concentrationof from 1 to 1000 nmol/ml, more preferably from 1 to 100 nmol/ml. In thecase of antibodies, the final concentration used is typically from 100to 500 μg/ml, more preferably from 200 to 300 μg/ml.

[0231] DNA Binding

[0232] When released from the nuclear environment, nucleic acid such asDNA can cause inflammation in the surrounding tissues. Although severalDNA-binding proteins are known, the proteins or receptors that involvein the clearance of DNA from the lung and other tissues are not clearlyestablished. The present inventors have discovered that SP-D bindseffectively to DNA (and total RNA) of bacterial, bacteriophage andchromosomal origin.

[0233] Electron microscopy shows that recombinant fragment ofrSPD(n/CRD) (rSPD(n/CRD)) can efficiently bind DNA. Although not wishingto be bound by theory, the inventors have determined that nucleotides,the building blocks of DNA, can compete with mannan for binding torSPD(n/CRD), suggesting that this protein can bind DNA via itscarbohydrate binding activity. In addition, the presence of a shortcollagen-like fragment in this recombinant fragment may also provideadded ability for it to bind DNA, effectively. Furthermore, rSPD(n/CRD)competes with propidium iodide for the binding of DNA on apoptotic cellsindicating that the protein binds to the DNA found on the surface ofthese cells. Recognition of DNA on the surface is likely to be one ofthe important modes of clearing the apoptotic cells from the lung.

[0234] Several applications for SP-D and fragments thereof, especiallyrSPD(n/CRD), are mediated via the DNA-binding properties of thesemolecules. For example:

[0235] 1) Several diseases, many of which involve chronic bacterial,fungal and viral infection result in the accumulation of free nucleicacid in lung. Cystic fibrosis is one such disease, and persistentinfection in lung occurs only after bacterial bio-film formation, whichrequires extracellular free DNA. Since native SP-D and rSPD(n/CRD) bindfree DNA, these proteins enhance the clearance of DNA and minimizebio-film formation. The rSPD(n/CRD) also opsonizes many pulmonarymicrobes, and acts potently to reduce or eliminate pathogen colonizationin cystic fibrosis patients.

[0236] 2) SP-D and rSPD(n/CRD) can bind to DNA. Therefore, cells thatcontain DNA on their surfaces, such as apoptotic and necrotic cells, arealso recognized by SP-D and rSPD(n/CRD). These facts provide amechanistic explanation by which the inflammation-causing cells arerecognized by the rSPD(n/CRD) for phagocyte-mediated clearance, in vivo.SP-D and fragments thereof can be used to treat lung diseases andconditions such as COPD, chronic inflammation and chronic asthma.

[0237] 3) Free DNA or DNA debris from necrotic and apoptotic cells canact as an autoantigen. Therefore, rSPD(n/CRD), in producing clearance offree DNA (or RNA) helps in reducing autoantibody production inautoimmune diseases.

[0238] Inflammatory Diseases

[0239] The term “inflammation” is used here to refer to the response ofliving tissue to damage. An rSPD(n/CRD) polypeptide, fragment,homologue, variant or derivative thereof may be used to treatinflammation, including an inflammatory disease, by administering anindividual suffering from such a disease with the rSPD(n/CRD)polypeptide, fragment, homologue, variant or derivative thereof.

[0240] The causes of inflammation include physical damage, chemicalsubstances, microorganisms or other agents. In a preferred embodiment,the rSPD(n/CRD) polypeptide, fragment, homologue, variant or derivativethereof is used to treat an inflammatory disease, although it may beused to treat any inflammation resulting from any cause.

[0241] The inflammatory response consists of changes in blood flow,increased permeability of blood vessels and escape of cells from theblood into the tissues. Acute inflammation is short-lasting, lastingonly a few days. If it is longer lasting however, then it is referred toas chronic inflammation. Various examples of acute inflammation that youmay be aware of are sore throat, reactions in the skin to a scratch or aburn or insect bite, and acute hepatitis and so on. However, there areoccasional historical exceptions such as pneumonia, inflammation of thelung rather than pneumonitis and pleurisy, inflammation of the pleura,rather than pleuritis.

[0242] Microbial Infections

[0243] Viruses lead to death of individual cells by intracellularmultiplication. Bacteria release specific exotoxins—chemicalssynthesised by them which specifically initiate inflammation orendotoxins, which are associated with their cell walls. Additionally,some organisms cause immunologically-mediated inflammation throughhypersensitivity reactions. Parasitic infections and tuberculousinflammation are instances where hypersensitivity is important.

[0244] Hypersensitivity Reactions

[0245] A hypersensitivity reaction occurs when an altered state ofimmunological responsiveness causes an inappropriate or excessive immunereaction which damages the tissues. All have cellular or chemicalmediators similar to those involved in inflammation.

[0246] Physical Agents

[0247] Tissue damage leading to inflammation may occur through physicaltrauma, ultraviolet or other ionising radiation, bums or excessivecooling (“frostbite”).

[0248] Irritant and Corrosive Chemicals

[0249] Corrosive chemicals (acids, alkalis, oxidising agents) provokeinflammation through gross tissue damage. However, infecting agents mayrelease specific chemical irritants which lead directly to inflammation.

[0250] Inflammation typically involves at least one of the followingfeatures:

[0251] Tissue necrosis: Death of tissues from lack of oxygen ornutrients resulting from inadequate blood flow (infarction) is a potentinflammatory stimulus. The edge of a recent infarct often shows an acuteinflammatory response. Redness (rubor): An acutely inflamed tissueappears red, for example skin affected by sunburn, cellulitis due tobacterial infection or acute conjunctivitis. This is due to dilatationof small blood vessels within the damaged area. Heat (calor): Increasein temperature is seen only in peripheral parts of the body, such as theskin. It is due to increased blood flow (hyperaemia) through the region,resulting in vascular dilatation and the delivery of warm blood to thearea. Systemic fever, which results from some of the chemical mediatorsof inflammation, also contributes to the local temperature. Swelling(tumor): Swelling results from oedema, the accumulation of fluid in theextra vascular space as part of the fluid exudate, and to a much lesserextent, from the physical mass of the inflammatory cells migrating intothe area. Pain (dolor): For the patient, pain is one of the best knownfeatures of acute inflammation. It results partly from the stretchingand distortion of tissues due to inflammatory oedema and, in particular,from pus under pressure in an abscess cavity. Some of the chemicalmediators of acute inflammation, including bradykinin, theprostaglandins and serotonin, are known to induce pain. Loss offunction: Movement of an inflamed area is consciously and reflexlyinhibited by pain, while severe swelling may physically immobilise thetissues.

[0252] Thus, we provide for the alleviation of a symptom ofinflammation, including a symptom as listed above, by the administrationof an rSPD(n/CRD) polypeptide, fragment, homologue, variant orderivative thereof to an individual suffering from inflammation or aninflammatory disease.

[0253] Inflammatory Lung Disease

[0254] In a highly preferred embodiment, a rSPD(n/CRD) polypeptide,fragment, homologue, variant or derivative thereof is used to treat aninflammatory lung disease.

[0255] As the term is used here, “inflammatory lung disease” should betaken to mean any disease or condition which involves inflammation ofthe lung, or any portion of the lung or associated systems such as theairway, trachea etc. The inflammatory lung disease may be acute orchronic, and may a result of or be accompanied by some other disease orsyndrome. In a preferred embodiment, the inflammatory lung diseasecomprises a chronic inflammatory lung disease.

[0256] Examples of inflammatory lung diseases include neonatal chroniclung disease, neonatal respiratory distress syndrome (RDS), adultrespiratory distress syndrome, chronic obstructive airways disease(COPD), asthma, cystic fibrosis, pulmonary fibrosis, emphysema,interstitial inflammatory lung disease, sarcoidosis, pneumonia, chronicinflammatory lung disease, neonatal chronic inflammatory lung disease.

[0257] Individuals suffering from such diseases may be treated by, ortheir symptoms relieved, by administration of a rSPD(n/CRD) polypeptide,fragment, homologue, variant or derivative thereof to the individual.Preferably, such treatment results in the clearance of alveolarmacrophages, preferably apoptotic alveolar macrophages. Treatment mayalso further or in the alternative result in clearance of necroticmacrophages. Necrotic and apoptotic cells may be detected anddistinguished by the assays as set out below.

[0258] Neonatal Chronic Lung Disease

[0259] Neonatal chronic lung disease is a multifactorial conditionassociated with recurrent inflammation and infection after prematurebirth. There are a number of factors carrying an increased risk ofdeveloping chronic lung disease including premature birth, prolongedventilation, recurrent infection in the neonatal period, surfactantdeficiency and increased inflammatory markers in the neonatal period,including the number of alveolar macrophages in tracheal suctionsamples. Premature infants are known to be deficient in surfactantproteins including SP-D.

[0260] Human neonatal chronic lung disease arises out of inflammatorydamage and oxidative stress after prolonged assisted ventilation,typically after premature birth. Survival after premature birth hasincreased dramatically since the advent of artificial surfactantreplacement therapy for the treatment of neonatal respiratory distresssyndrome, but up to 40% of infants surviving after birth at less than 28weeks gestation develop chronic inflammatory lung disease. Currentsurfactant replacement strategies do not include surfactant protein D.Lung damage in this setting is mediated by activated alveolarmacrophages. SP-D deficient mice provide a model for macrophage mediatedinflammation in the lung characterised by the presence of large numbersof activated alveolar macrophages.

[0261] We demonstrate in the Examples that intranasal administration ofrSP-D(N/CRD) to SP-D deficient mice modulates the inflammatory processin the lungs by decreasing the number of activated macrophages andaltering their phenotype. The results demonstrate a potentanti-inflammatory property of rSP-D(N/CRD) and indicate a directclinical application of rSP-D(N/CRD) in the prevention of chronicinflammatory lung disease in neonates by direct administration or byincluding rSP-D(N/CRD) in the formulation of current surfactanttherapies. Nucleic acids encoding rSPD(n/CRD), as well as fragments,homologues, variants and derivatives may also be employed. Chronic lunginflammation causing emphysema (e.g. in smokers, deficient in naturallevels of SP-D) may also be treatable by administration of rSP-D(N/CRD).

[0262] We therefore provide for the treatment of neonatal chronic lungdisease, or any symptom of such a disease, by the administration of anrSPD(n/CRD) polypeptide, fragment, homologue, variant or derivativethereof.

[0263] Surfactant Replacement Therapy

[0264] This section describes Surfactant Replacement Therapy, for whichthe rSPD(n/CRD) polypeptide, nucleic acid, or a fragment, homologue,variant or derivative thereof disclosed here are suitably used. Inparticular, the rSPD(n/CRD) polypeptide, nucleic acid, or a fragment,homologue, variant or derivative thereof may be employed for surfactantreplacement therapy in the neonate.

[0265] Surfactant Replacement Therapy is described in detail in Respir.Care, 1994, 39(8):824-829, from which this section is derived.Guidelines for Surfactant Replacement Therapy have been issued, forexample, the AARC Clinical Practice Guidelines. A clinician followingsuch guidelines will be able to administer rSPD(n/CRD) polypeptide,nucleic acid, or a fragment, homologue, variant or derivative thereof aspart of a programme of surfactant replacement therapy.

[0266] Natural, endogenous surfactant is a compound composed ofphospholipids, neutral lipids, and proteins (1-5) that forms a layerbetween the alveolar surface and the alveolar gas and reduces alveolarcollapse by decreasing surface tension within the alveoli. (3-5)Surfactant deficiency is almost always associated with the formation ofhyaline membranes in the immature lung and the onset of respiratorydistress syndrome (RDS)—a major cause of morbidity and mortality inpremature infants. (3) Without surfactant, alveoli may never inflate ormay collapse on expiration and require inordinate force to re-expand oninspiration, leading to the development of RDS. (3,5) The incidence ofRDS is related more to lung immaturity than to gestational age. (6)However, in general, the more premature the infant, the less thesurfactant production and the higher the probability for RDS. (4,6)Direct tracheal instillation of surfactant has been shown to reducemortality and morbidity in infants with RDS. (7-25)

[0267] Surfactant can be extracted from animal lung lavage and fromhuman amniotic fluid or produced from synthetic materials. According tothe methods and compositions described here, rSPD(n/CRD) polypeptide,nucleic acid, or a fragment, homologue, variant or derivative thereofmay be used for Surfactant Replacement Therapy, particularly in theneonate. rSPD(n/CRD) polypeptide, nucleic acid, or a fragment,homologue, variant or derivative thereof may be used alone, or incombination with known surfactant replacement therapies.

[0268] Two basic strategies for surfactant replacement have emerged: (1)prophylactic or preventive treatment in which surfactant is administeredat the time of birth or shortly thereafter to infants who are at highrisk for developing RDS and (2) rescue or therapeutic treatment in whichsurfactant is administered after the initiation of mechanicalventilation in infants with clinically confirmed RDS. (2,10-12,26,27).

[0269] Surfactant Replacement Therapy is typically administered bytrained personnel in the delivery room or in a neonatal intensive careunit.

[0270] Indications for Surfactant Replacement Therapy

[0271] 4.1 Prophylactic administration may be indicated in 4.1.1 infantsat high risk of developing RDS because of short gestation (<32 weeks)(8,10-12,21,25-29) or low birth weight (<1,300 g), (21-25,28) whichstrongly suggest lung immaturity. 4.1.2 infants in whom there islaboratory evidence of surfactant deficiency such aslecithin/sphingomyelin ratio less than 2:1, (11,14,28,30,31) bubblestability test indicating lung immaturity, (15,32) or the absence ofphosphatidylglycerol. (11,14,22-24,28,30) 4.2 Rescue or therapeuticadministration is indicated in preterm or full-term infants 4.2.1 whorequire endotracheal intubation and mechanical ventilation because of4.2.1.1 increased work of breathing as indicated by an increase inrespiratory rate, substernal and suprasternal retractions, grunting, andnasal flaring.(8,11,14-16,29,33-35) 4.2.1.2 increasing oxygenrequirements as indicated by pale or cyanotic skin color, agitation, anddecreases in PaO2, SaO2, or SpO2 mandating an increase in FIO2 above 0.(4011,12,15,26,33,36-38) and 4.2.2 have clinical evidence of RDS,(13,39) including 4.2.2.1 chest radiograph characteristic of RDS,(8,11-16,33,34,36,37,40-42) 4.2.2.2 mean airway pressure greater than 7cm H2O to maintain an adequate PaO2, SaO2, or SpO2. (11,14,15,26,43)

[0272] Contraindications

[0273] Surfactant Replacement Therapy is contraindicated for thefollowing conditions: 5.1 the presence of congenital anomaliesincompatible with life beyond the neonatal period,(8,14,15,26,28,29,31,33,36,41,44) 5.2 respiratory distress in infantswith laboratory evidence of lung maturity. (9,14,27-29,33,36,41)

[0274] Hazards and complications arising from Surfactant ReplacementTherapy include: 6.1 Procedural complications resulting from theadministration of surfactant include 6.1.1 plugging of endotracheal tube(ETT) by surfactant; (2) 6.1.2 hemoglobin desaturation and increasedneed for supplemental O2; (11,33,41) 6.1.3 bradycardia due to hypoxia;(9,33,41,45) 6.1.4 tachycardia due to agitation, with reflux ofsurfactant into the ETT; (34,41) 6.1.5 pharyngeal deposition ofsurfactant; 6.1.6 administration of surfactant to only one lung; 6.1.7administration of suboptimal dose secondary to miscalculation or errorin reconstitution. 6.2 Physiologic complications of surfactantreplacement therapy include 6.2.1 apnea, (7,13,15) 6.2.2 pulmonaryhemorrhage, (12,15,18,32,34,38,46,47) 6.2.3 mucus plugs, (48) 6.2.4increased necessity for treatment for PDA, (18,29,30) 6.2.5 marginalincrease in retinopathy of prematurity, (11) 6.2.6 barotrauma resultingfrom increase in lung compliance following surfactant replacement andfailure to change ventilator settings accordingly. (30,49)

[0275] Surfactant administered prophylactically may be given to someinfants in whom RDS would not have developed. (10,12,26,33) Whensurfactant is administered prophylactically in the delivery room, ETTplacement may not have been verified by chest radiograph resulting inthe inadvertent administration to only one lung or to the stomach. (26)Prophylactic surfactant administration may delay patient stabilization.(26) Atelectasis and lung injury may occur prior to therapeuticadministration. (26,33) Tracheal suctioning should be avoided followingsurfactant administration. (9,11,13,14,27,33,38,44,50) Not all infantswho are treated with a single dose of surfactant experience a positiveresponse 39 or the response may be transient. Positioning recommendedfor surfactant administration may further compromise the unstableinfant. (9,11,12,14,16,28,33,38-40)

[0276] A clinician may undertake an assessment of need to determine thatvalid indications are present. The following are typically done forthis: 8.1 Assess lung immaturity prior to prophylactic administration ofsurfactant by gestational age and birth weight and/or by laboratoryevaluation of tracheal or gastric aspirate. 8.2 Establish the diagnosisof RDS by chest radiographic criteria and the requirement for mechanicalventilation in the presence of short gestation and/or low birth weight.

[0277] Assessment of outcome is conducted according to the following:9.1 Reduction in FIO2 requirement (12,33,34,36-39,41,44) 9.2 Reductionin work of breathing (51) 9.3 Improvement in lung volumes and lungfields as indicated by chest radiograph (13,16,33,40) 9.4 Improvement inpulmonary mechanics (e.g., compliance, airways resistance, VT, VE,transpulmonary pressure) and lung volume (i.e., FRC) (42,43,50,52-59)9.5 Reduction in ventilator requirements (PIP, PEEP, Paw)(2,8,9,12,13,27,30,33,36-39,41,44,50,52) 9.6 Improvement in ratio ofarterial to alveolar PO2 (a/A PO2), oxygen index.(13,16,28,30,33,34,37-41,44)

[0278] Resources

[0279] Administration procedures recommended for specific preparationsof surfactant should be adhered to. 10.1 Equipment(10-14,16,26-28,33,34,39,40,50,60) 10.1.1 Administration equipment10.1.1.1 Syringe containing the ordered dose of surfactant, warmed toroom temperature (11,12,16,38,40) 10.1.1.2 5-Fr feeding tube orcatheter, or endotracheal tube connector with delivery port 10.1.1.3Mechanical ventilator or manual ventilator (resuscitation bag)(8,16,33,36,38-40,44,50,52) 10.1.2 Resuscitation equipment 10.1.2.1Laryngoscope and endotracheal tube (10-12,14,16,26,38) 10.1.2.2 Manualresuscitation bag (9-12,16,26-28,36,39,40,50) and airway manometer10.1.2.3 Blended oxygen source (9,16,28,44) 10.1.2.4 Suction equipment(i.e., catheters, sterile gloves, collecting bottle and tubing, andvacuum generator) (9,33,50,60) 10.1.2.5 Radiant warmer ready for use10.1.3 Monitoring equipment 10.1.3.1 Neonatal tidal volume monitor ifavailable (50) 10.1.3.2 Airway pressure monitor 10.1.3.3 Pulse oximeteror transcutaneous PCO2 monitor (11,26,34,39-41,52) 10.1.3.4Cardiorespiratory monitor 10.2 Personnel—Surfactant replacement therapyshould be performed under the direction of a physician by credentialedpersonnel (e.g., CRTT, RRT, RN) who competently demonstrate 10.2.1proper use, understanding, and mastery of the equipment and technicalaspects of surfactant replacement therapy; 10.2.2 comprehensiveknowledge and understanding of neonatal ventilator management andpulmonary anatomy and pathophysiology; 10.2.3 neonatal patientassessment skills, including the ability to recognize and respond toadverse reactions and/or complications of the procedure; 10.2.4knowledge and understanding of the patient's history and clinicalcondition; 10.2.5 knowledge and understanding of airway management,10.2.6 ability to interpret monitored and measured blood gas variablesand vital signs; 10.2.7 proper use, understanding, and mastery ofemergency resuscitation equipment and procedures; 10.2.8 ability toevaluate and document outcome (Section 9.0); 10.2.9 understanding andproper application of Universal Precautions.

[0280] Monitoring

[0281] The following should be monitored as part of surfactantreplacement therapy. 11.1 Variables to be monitored during surfactantadministration 11.1.1 Proper placement and position of delivery device11.1.2 FIO2 and ventilator settings (8,9,11,13-15,27-29,33,36,38,44)11.1.3 Reflux of surfactant into ETT (34,41) 11.1.4 position of patient(i.e., head direction) (9,11,33) 11.1.5 Chest-wall movement (6 1) 11.1.6Oxygen saturation by pulse oximetry (11,26,34,39-41,52) 11.1.7 Heartrate, respirations, chest expansion, skin color, and vigor(16,26,27,34,41,45,52) 11.2 Variables to be monitored after surfactantadministration 11.2.1 Invasive and noninvasive measurements of arterialblood gases (8,9,11,12-16,26-29,33,36,38,39,41,44) 11.2.2 Chestradiograph (11-16,28,36,38-40,44) 11.2.3 Ventilator settings (PIP, PEEP,Paw) and FIO2 (8,9,11,13-16,28,29,33,36,38) 11.2.4 Pulmonary mechanicsand volumes 11.2.5 Heart rate, respirations, chest expansion, skincolor, and vigor (16,26,27,34,41,45,52) 11.2.6 Breath sounds (11,38)11.2.7 Blood pressure 1. (3,16,33,40,44,45)

[0282] Frequency

[0283] Repeat doses of surfactant are contingent upon the continueddiagnosis of RDS. The frequency with which surfactant replacement isperformed should depend upon the clinical status of the patient and theindication for performing the procedure. Additional doses of surfactant,given at 6- to 24-hour intervals, may be indicated in infants whoexperience increasing ventilator requirements or whose conditions failto improve after the initial dose. (7,9,11,12,14,15,26,30,34,37,39,52)

[0284] Infection Control

[0285] 13.1 Universal Precautions (62) should be implemented. 13.2Aseptic technique should be practiced. 13.3 Appropriate infectioncontrol guidelines for the patient should be posted and followed.

[0286] Cystic Fibrosis

[0287] We show in the Examples that rSPD(n/CRD) polypeptide is capableof reducing alveolar macrophage number. Accordingly, rSPD(n/CRD)polypeptide, nucleic acid, or a fragment, homologue, variant orderivative thereof may be administered in lung diseases in whichalveolar macrophages play a role in the inflammatory process, forexample, cystic fibrosis, asthma and emphysema. rSP-D (N/CRD) also hastherapeutic properties in macrophage mediated inflammatory processes atother sites, e.g. in atherosclerosis.

[0288] Cystic fibrosis is a genetic disease affecting approximately bothchildren and adults.

[0289] Cystic fibrosis causes the body to produce an abnormally thick,sticky mucus, due to the faulty transport of sodium and chloride (salt)within cells lining organs such as the lungs and pancreas, to theirouter surfaces. The thick cystic fibrosis mucus also obstructs thepancreas, preventing enzymes from reaching the intestines to help breakdown and digest food.

[0290] Cystic fibrosis has a variety of symptoms. The most common are:very salty-tasting skin; persistent coughing, wheezing or pneumonia;excessive appetite but poor weight gain; and bulky stools. The sweattest is the standard diagnostic test for cystic fibrosis, which measuresthe amount of salt in the sweat. A high salt level indicates that aperson has cystic fibrosis.

[0291] The treatment of cystic fibrosis depends upon the stage of thedisease and which organs are involved. One means of treatment, chestphysical therapy, requires vigorous percussion (by using cupped hands)on the back and chest to dislodge the thick mucus from the lungs.Antibiotics are also used to treat lung infections and are administeredintravenously, via pills, and/or medicated vapors which are inhaled toopen up clogged airways. When cystic fibrosis affects the digestivesystem, the body does not absorb enough nutrients. Therefore, peoplewith cystic fibrosis may need to eat an enriched diet and need to takeboth replacement vitamins and enzymes.

[0292] According to the methods and compositions described here, asymptom of a patient suffering from cystic fibrosis is alleviated byadministration of rSPD(n/CRD) polypeptide, fragment, homologue, variantor derivative thereof. The symptom of cystic fibrosis preferablyincludes any of the symptoms listed above. Preferably, a patientsuffering from cystic fibrosis is treated by administration of such apolypeptide, homologue, fragment or derivative.

[0293] Allergies

[0294] Existing treatments for allergies typically involve the long-termuse of steroids to depress the immune system. There are undesirable sideeffects with long-term steroid therapy. We demonstrate that rSPD(n/CRD)polypeptide, nucleic acid, or a fragment, homologue, variant orderivative thereof may be used to alleviate the symptoms of allergy, orto treat allergy.

[0295] Treatment with rSPD(n/CRD) appears to have no ill effects in miceand is unlikely to generate unwanted side-effects in humans. A shorttreatment course with rSPD(n/CRD) can reverse the underlying allergicsensitisation leading to effective immunotherapy. This is a significantimprovement on existing desensitisation immunotherapy protocols whichtypically involve a series of injections of allergen over a period ofyears.

[0296] Allergies suitable for treatment with rSPD(n/CRD) polypeptide,nucleic acid, or a fragment, homologue, variant or derivative thereofinclude a seasonal respiratory allergy, allergic rhinitis, hayfever,nonallergic rhinitis, vasomotor rhinitis, irritant rhinitis, an allergyagainst grass pollens, tree pollens or animal danders, an allergyassociated with allergic asthma, and food allergies. In particular, andas described elsewhere, rSPD(n/CRD) polypeptide, nucleic acid, or afragment, homologue, variant or derivative thereof may be used to treatallergies to house dust mite (Dermatophagoides spp), preferablyDermatophagoides pteronyssinus or Dermatophagoides farinae, or to fungior fungal spores, preferably Aspergillus fumigatus. Preferably, theallergens are comprised in faeces of Dermatophagoides spp.

[0297] Asthma

[0298] Asthma is a respiratory condition characterized by episodes ofairflow obstruction in the bronchial tubes. Symptoms caused by thisobstruction include coughing, chest tightness, wheezing and shortness ofbreath. Although problems are often separated by symptom-free periods,asthma is a chronic illness.

[0299] A wide variety of “triggers” may initiate an episode of asthma.The most common triggers are allergens, aspirin and tartrazine,irritants, food additives and preservatives, viral respiratoryinfections and physical exertion.

[0300] Allergens are substances to which susceptible individuals maybecome allergic. They are a major source of problems in children andadults. Common allergens include plant pollen (tree, grass and weed),animal dander, house dust mites, molds and certain foods. When anallergic individual comes in contact with one of these allergens, aseries of events causes the body to release certain chemicals(mediators), which then trigger asthma.

[0301] Ten to twenty percent of asthmatics experience a significantdecrease in their lung function after taking aspirin; similar reactionscan occur with a related group of medications called nonsteroidalanti-inflammatory agents and with tartrazine (yellow food dye #5). Coldair, smoke, industrial chemicals, perfume, paint and gasoline fumes areall examples of irritants that can provoke asthma, probably bystimulating irritant receptors in the respiratory tract. Thesereceptors, in turn, cause the muscles surrounding the airway toconstrict, resulting in an asthma attack. Rarely, food additives such assulphites can trigger asthma. Viral respiratory infections are theleading cause of acute asthma attacks.

[0302] During an asthma attach, an asthmatic's hyperactive bronchialtubes narrow in response to certain triggers. During an attack, musclessurrounding the bronchial tubes contract, narrowing the air passages.Inflammation also occurs along the lining of the airways which producesswelling and further reduction of airway size. In addition, mucus glandsalong the inside of the air passages produce excess mucus whichaccumulates in the already narrowed air passages. The end result is thatbreathing, especially exhaling, becomes extremely difficult. Air becomestrapped behind the narrowed bronchial passages and there is a decreasein the oxygen available to the body.

[0303] Bronchodilators are the most commonly prescribed drugs to treatasthma. They relax the muscles surrounding the airways, resulting indilation of the bronchial tubes. Bronchodilators may be inhaled, takenorally or injected. Cold, dry air, for example, inhaled during exercisethrough the mouth can also trigger asthma.

[0304] Current drugs used for treating the symptoms of asthma includelong-acting theophyllines, inhaled or oral beta agonists, cromolyn andinhaled or oral steroids. For allergic asthmatics, immunotherapy(allergy shots) may offer relief from allergens that cannot be avoided.Immunotherapy increases a patient's tolerance to the allergens thatprompt asthma symptoms.

[0305] Allergic Asthma

[0306] Asthma may also be triggered by exposure to allergens (“allergicasthma”).

[0307] Allergic asthma is characterised by periods of airwayhyperresponsiveness (AHR.) that occur in early phase and late phasehypersensitivity reactions in response to allergen provocation. Theearly phase response is mediated by the degranulation of airway mastcells with the release of histamine and other bronchoconstrictors. Thelate phase response begins after the influx of inflammatory cells suchas eosinophils and lymphocytes and the subsequent release ofinflammatory mediators and cytokines, which lead to chronic AHR.

[0308] House dust mite is one of the leading causes of allergic asthma(Platts-Mills, T. A. and M. D. Chapman, 1987, Dust mites: immunology,allergic disease, and environmental control, J. Allergy Clin. Immunol.80:755-75; Platts-Mills, T. A., E. B. Mitchell, M. D. Chapman, and P. W.Heymann, 1987, Dust mite allergy: its clinical significance, Hosp.Pract. (Off Ed) 22:91-3, 97-100; Pollart, S. M., M. D. Chapman, and T.A. Platts-Mills, 1987, House dust sensitivity and environmental control,Prim. Care. 14:591-603).

[0309] An rSPD(n/CRD) polypeptide, fragment, homologue, variant orderivative thereof for example as described here, may be used to treatallergic asthma. Preferably, the rSPD(n/CRD) polypeptide, fragment,homologue, variant or derivative thereof is employed to treat allergy,or allergic asthma, caused by house dust mite.

[0310] House dust mite refers to any organisms of Dermatophagoides spp.Preferably, the Dermatophagoides organism is Dermatophagoidespteronyssinus or Dermatophagoides farinae. Allergic asthma may alsodevelop as a result of exposure of an individual to fungi or fungalspores, and the methods and compositions described here include thetreatment of such asthma or allergy, including allergy againstAspergillus fumigatus antigens.

[0311] Pulmonary Emphysema And Chronic Obstructive Pulmonary Disease

[0312] In a particular embodiment, an rSPD(n/CRD) polypeptide, fragment,homologue, variant or derivative thereof, for example as described here,is used to treat pulmonary emphysema or chronic obstructive pulmonarydisease.

[0313] Emphysema is a massive public health problem for which there areno satisfactory treatments. Cigarette smoke induces alveolar macrophageapoptosis in vitro (Aoshiba, K., J. Tamaoki, and A. Nagai, 2001, Acutecigarette smoke exposure induces apoptosis of alveolar macrophages, Am.J. Physiol. Lung Cell. Mol. Physiol. 281:L1392) and in vivo (Aoshiba etal., supra) and Majo et al. has recently reported that apoptosis in lungtissue samples from smokers showed a bilinear relationship with theamount smoked, increasing sharply in smokers with emphysema andconcludes that apoptosis might be one of the mechanisms of lungdestruction leading to the development of emphysema Majo, J., H. Ghezzo,and M. G. Cosio, 2001, Lymphocyte population and apoptosis in the lungsof smokers and their relation to emphysema, Eur. Respir. J. 17:946. Wepropose that SP-D is deficient in smokers, and that this promotesapoptosis and contribute to emphysema in this patient group. Wedemonstrate that rSP-D(N/CRD) administration to SP-D deficient micereduces apoptotic cell numbers; accordingly, rSP-D(N/CRD) therapy inhumans can inhibit a mechanism contributing to emphysema provoked bycigarette smoking.

[0314] Cytokines

[0315] In a further embodiment, the rSPD(n/CRD) polypeptide, nucleicacid, fragment, homologue, variant or derivative thereof is used tomodulate cytokine levels in an individual. Preferably, the level ofinflammatory cytokines is down-regulated. Examples of inflammatorycytokines include Granulocyte-Macrophage-Colony stimulating factor(GM-CSF), as well as any cytokine that mediates migration of alveolarmacrophages into the lung and act to increase cell proliferation.

[0316] Modulation of GM-CSF levels by administration of rSPD(n/CRD)polypeptide, nucleic acid, or a fragment, homologue, variant orderivative thereof is demonstrated in Example 9 (FIG. 14).

[0317] The term “cytokine” may be used to refer to any of a number ofsoluble molecules (e.g., glycoproteins) released by cells of the immunesystem, which act nonenzymatically through specific receptors toregulate immune responses. Cytokines resemble hormones in that they actat low concentrations bound with high affinity to a specific receptor.Preferably, the term “cytokine” refers to a diverse group of solubleproteins and peptides which act as humoral regulators at nano- topicomolar concentrations and which, either under normal or pathologicalconditions, modulate the functional activities of individual cells andtissues.

[0318] Particular examples of cytokines which are suitable for use inthe methods and compositions described include interleukins, lymphokine,interferon, Colony Stimulating Factors (CSFs) such as Granulocyte-ColonyStimulating Factor (G-CSF), Macrophage-Colony stimulating factor (M-CSF)and Granulocyte-Macrophage-Colony stimulating factor (GM-CSF), GSF,Platelet-Activating Factors (PAF), Tumor Necrosis Factor (TNF).

[0319] Thus, interleukins such as IL1, IL2 and IL4, as well asinterferons such as IFN-

, IFN-

and IFN-

are included. Tumour necrosis factors TNF-

(cachetin), TNF-

(lymphotoxin) may also be suitably employed.

[0320] Preferred cytokines are those which are capable of recruitingimmune responses, for example, stimulation of dendritic cell orcytotoxic T cell activity, or which are capable of recruitingmacrophages to the target site. In a highly preferred embodiment, thecytokine comprises IL-2, GM-CSF or GSF.

[0321] Apoptosis

[0322] According to the methods and compositions described here,administration of a rSPD(n/CRD) polypeptide, nucleic acid, or afragment, homologue, variant or derivative thereof to an individual iscapable of reducing the number of alveolar macrophages, in particularthe number of apoptotic alveolar macrophages, or necrotic alveolarmacrophages or both. Without seeming to be bound by a particular theory,we envisage that rSPD(n/CRD) polypeptide, nucleic acid, or a fragment,homologue, variant or derivative thereof achieves this by enhancing theclearance of apoptotic and/or necrotic macrophages for the body of theindividual.

[0323] Preferably, treatment with rSPD(n/CRD) polypeptide, nucleic acid,or a fragment, homologue, variant or derivative thereof results in least10% more clearance of apoptotic and/or necrotic macrophages, preferablyat least 20% more clearance. Most preferably, treatment with rSPD(n/CRD)polypeptide, nucleic acid, or a fragment, homologue, variant orderivative thereof results in at least 40%, 60%, 80% or more clearanceof apoptotic and/or necrotic macrophages.

[0324] Cell death can occur by either of two distinct mechanisms,necrosis or apoptosis. In addition, certain chemical compounds and cellsare said to be cytotoxic to the cell, that is, to cause its death.

[0325] “Necrosis” (also referred to as “accidental” cell death) refersto the pathological process which occurs when cells are exposed to aserious physical or chemical insult. Necrosis occurs when cells areexposed to extreme variance from physiological conditions (e.g.,hypothermia, hypoxia) which may result in damage to the plasma membrane.Under physiological conditions direct damage to the plasma membrane isevoked by agents like complement and lytic viruses. Necrosis begins withan impairment of the cell's ability to maintain homeostasis, leading toan influx of water and extracellular ions. Intracellular organelles,most notably the mitochondria, and the entire cell swell and rupture(cell lysis). Due to the ultimate breakdown of the plasma membrane, thecytoplasmic contents including lysosomal enzymes are released into theextracellular fluid. Therefore, in vivo, necrotic cell death is oftenassociated with extensive tissue damage resulting in an intenseinflammatory response.

[0326] “Apoptosis” (“normal” or “programmed” cell death) refers to thephysiological process by which unwanted or useless cells are eliminatedduring development and other normal biological processes. Apoptosis is amode of cell death that occurs under normal physiological conditions andthe cell is an active participant in its own demise (“cellularsuicide”). It is most often found during normal cell turnover and tissuehomeostasis, embryogenesis, induction and maintenance of immunetolerance, development of the nervous system and endocrine dependenttissue atrophy. Cells undergoing apoptosis show characteristicmorphological and biochemical features. These features include chromatinaggregation, nuclear and cytoplasmic condensation, partition ofcytoplasm and nucleus into membrane bound vesicles (apoptotic bodies)which contain ribosomes, morphologically intact mitochondria and nuclearmaterial. In vivo, these apoptotic bodies are rapidly recognised andphagocytized by either macrophages or adjacent epithelial cells. Due tothis efficient mechanism for the removal of apoptotic cells in vivo noinflammatory response is elicited. In vitro, the apoptotic bodies aswell as the remaining cell fragments ultimately swell and finally lyse.This terminal phase of in vitro cell death has been termed “secondarynecrosis”.

[0327] Table 1 summarises the various observable differences betweennecrosis and apoptosis. Any of these differences, alone or incombination, may be assayed in order to determine whether cell death isoccurring by apoptosis or by necrosis. TABLE 1 Differential features andsignificance of necrosis and apoptosis. Necrosis Apoptosis MorphologicalLoss of membrane Membrane blebbing, but no loss of integrity featuresintegrity Aggregation of chromatin at the nuclear Begins with swellingof membrane cytoplasm and Begins with shrinking of cytoplasm andmitochondria condensation of nucleus Ends with total cell lysis Endswith fragmentation of cell into smaller No vesicle formation, bodiescomplete lysis Formation of membrane bound vesicles (apoptoticDisintegration (swelling) bodies) of organelles Mitochondria becomeleaky due to pore formation involving proteins of the bcl-2 family.Biochemical Loss of regulation of ion Tightly regulated processinvolving activation and features homeostasis enzymatic steps No energyrequirement Energy (ATP)-dependent (active process, does not (passiveprocess, also occur at 4° C.) occurs at 4° C.) Non-random mono- andoligonucleosomal length Random digestion of fragmentation of DNA (Ladderpattern after DNA (smear of DNA agarose gel electrophoresis) afteragarose gel Prelytic DNA fragmentation Release of variouselectrophoresis) factors (cytochrome C, AIF) into cytoplasm by PostlyticDNA mitochondria fragmentation (=late Activation of caspase cascadeevent of death Alterations in membrane asymmetry (i.e., translocation ofphosphatidyl-serine from the cytoplasmic to the extracellular side ofthe membrane) Physiological Affects groups of Affects individual cellssignificance contiguous cells Induced by physiological stimuli (lack ofgrowth Evoked by non- factors, changes in hormonal environment)physiological Phagocytosis by adjacent cells or macrophages disturbancesNo inflammatory response (complement attack, lytic viruses, hypothermia,hypoxia, ischemica, metabolic poisons) Phagocytosis by macrophagesSignificant inflammatory response

[0328] Reference is made to the following documents, which describeapoptosis in detail, as well as various assays for measuring cell deathby apoptosis: Schwartzman, R. A. and Cidlowski, J. A. (1993). EndocrineRev. 14, 133; Vermes, I. and Haanan, C. (1994). Adv. Clin. Chem. 31,177; Berke, G. (1991). Immunol. Today 12, 396; Krähenbühl, O. andTschopp, J. (1991). Immunol. Today 12, 399; Van Furth, R. and Van Zwet,T. L. (1988). J. Immunol; Methods 108, 45. Cohen, J. J. (1993)Apoptosis. Immunol. Today 14, 126; Savill, J. S. et al. (1989). J. Clin.Invest. 83, 865; Wyllie, A. H. (1980). Nature 284, 555; Leist, M. et al.(1994) Biochemica No. 3, 18-20; Fraser, A. and Evan, G. (1996) Cell 85,781-784; Duke, R. C. (1983). Proc. Natl. Acad. Sci. USA 80, 6361; Duke,R. C. & Cohen, J. J. (1986). Lymphokine Res. 5, 289; Trauth, B. C. etal. (1994) Eur. J. Cell. Biol. 63, 32, Suppl 40; Matzinger, P. (1991).J. Immunol; Methods 145, 185; Kaeck, M. R. (1993); Anal. Biochem. 208,393; Prigent, P. et al. (1993). J. Immunol; Methods 160, 139; Huang, P.& Plunkett, W. (1992); Anal. Biochem. 207,163 Bortner, C. D. et al.(1995) Trends Cell Biol. 5, 21; Gold, R. et al. (1994); Lab. Invest. 71,219.

[0329] Apoptosis and cell mediated cytotoxicity are characterised bycleavage of the genomic DNA into discrete fragments prior to membranedisintegration. Accordingly, apoptosis may be assayed by measuring DNAfragmentation, for example, by observing the presence of DNA ladders.DNA fragments may be assayed, for example, as “ladders” (with the 180 bpmultiples as “rungs” of the ladder) derived from populations of cells,or by quantification of histone complexed DNA fragments via, forexample, ELISA. Such an assay relies on an one-step sandwich immunoassayto detect nucleosomes. The procedure involves pelleting cells bycentrifugation and discarding the supernatant (which contains DNA fromnecrotic cells that leaked through the membrane during incubation).Cells are resuspended and incubated in lysis buffer. After lysis, intactnuclei are pelleted by centrifugation. An aliquot of the supernatant istransferred to a streptavidin-coated well of a microtiter plate, andnucleosomes in the supernatant are bound with two monoclonal antibodies,anti-histone (biotin-labelled) and anti-DNA (peroxidase-conjugated).Antibody-nucleosome complexes are bound to the microtiter plate by thestreptavidin. The immobilised antibody-histone complexes are washedthree times to remove cell components that are not immuno-reactive, andthe sample is incubated with peroxidase substrate (ABTS®). The amount ofcolored product (and thus, of immobilized anti-body-histone complexes)is then determined spectrophotometrically.

[0330] Several proteases are involved in the early stages of apoptosis.Apoptosis may therefore also be assayed by detecting the presence of, inaddition to, or instead of assaying the activity of, apoptosis-inducedproteases such as caspases, e.g., caspase 3. Caspase activation can beanalyzed in different ways, for example, by an in vitro enzyme assay of,for example, cellular lysates by capturing of the caspase and measuringproteolytic cleavage of a suitable substrate. Furthermore, caspases maybe assayed by detection of cleavage of an in vivo caspase substrate suchas PARP (Poly-ADP-Ribose-Polymer-ase). Cleaved fragments of PARP may bedetected with a suitable antibody such as an anti PARP antibody.Protease assays and DNA fragmentation assays are especially suitable forassaying apoptosis in cell populations.

[0331] Methods for studying apoptosis in individual cells are alsoavailable, such as ISNT and TUNEL enzymatic labeling assays. As notedabove, extensive DNA degradation is a characteristic event which oftenoccurs in the early stages of apoptosis. Cleavage of the DNA yieldsdouble-stranded, low molecular weight DNA fragments (mono- andoligonucleosomes) as well as single strand breaks (“nicks”) in highmolecular weight-DNA. In TUNEL, such DNA strand breaks are detected byenzymatic labeling of the free 3′-OH termini with suitable modifiednucleotides (such as X-dUTP, X=biotin, DIG or fluorescein). Suitablelabeling enzymes include DNA polymerase (nick translation) in ISNT (“insitu nick translation”) and terminal deoxynucleotidyl transferase (endlabeling) in TUNEL (“TdT-mediated X-dUTP nick end labeling”; Huang, P. &Plunkett, W., 1992, Anal. Biochem. 207, 163; Bortner, C. D. et al.,1995, Trends Cell Biol. 5, 21).

[0332] Apoptosis may also be assayed by measuring membrane alterations,including: loss of terminal sialic acid residues from the side chains ofcell surface glycoproteins, exposing new sugar residues; emergence ofsurface glycoproteins that may serve as receptors formacrophage-secreted adhesive molecules such as thrombospondin; and lossof asymmetry in cell membrane phospholipids, altering both thehydrophobicity and charge of the membrane surface. In particular, thehuman anticoagulant annexin V is a 35-36 kilodalton, Ca2+-dependentphospholipid-binding protein that has a high affinity forphosphatidylserine (PS). In normal viable cells, PS is located on thecytoplasmic surface of the cell membrane. However, in apoptotic cells,PS is translocated from the inner to the outer leaflet of the plasmamembrane, thus exposing PS to the external cellular environment. AnnexinV may therefore be used to detect phos-phatidylserine asymmetricallyexposed on the surface of apoptotic cells (Homburg, C. H. E. et al.1995, Blood 85, 532; Verhoven, B. et al., 1995, J. Exp. Med. 182, 1597).Furthermore, DNA stains such as DAPI, ethidium bromide and propidiumiodide, etc may be used for differential staining to distinguish viableand non-viable cells. Profiles of DNA content may also be used; thus,permeabilized apoptotic cells leak low molecular weight DNA, anddetection of “sub-G 1 peaks”, or “A 0” cells (cells with lower DNAstaining than that of G 1 cells) may be detected by, for example, flowcytometry. Morphological changes characteristic of apoptosis may also bedetected in this manner.

[0333] Detection of apoptosis-related proteins such as ced-3, ced-4,ced-9 (Ellis, H. M. and Horvitz, H. R., 1986, Cell 44, 817-829; Yuan, J.Y. and Horvitz, H. R., 1990, Dev. Biol. 138, 33-41; Hentgartner, M. O.,Ellis, R. E. and Horvitz, H. R., 1992, Nature 356, 494-499.),Fas(CD95/Apo-1; Enari et al., 1996, Nature 380, 723-726), Bcl-2 (Baffy,G. et al., 1993, J. Biol. Chem. 268, 6511-6519; Miyashita, T. and Reed,J. C., 1993, Blood 81, 151-157; Oltvai, Z. N., Milliman, C. L. andKorsmeyer, S. J., 1993, Cell 74, 609-619), p53 (Yonish-Rouach, E. etal., 1991, Nature 352, 345-347), etc by the use of antibodies may alsobe used to assay apoptosis.

[0334] Other Indications

[0335] The findings shown in the Examples support a therapeutic role forrSP-D N/CRD) in diseases in which the defective clearance or regulationof apoptotic and necrotic alveolar macrophages contributes to chronicpulmonary inflammation. Examples of such indications are discussedabove, in particular, the setting of relative SP-D deficiency such aschronic lung disease of the premature newborn and in smokers (who havedecreased SP-D levels) at risk of developing emphysema due to chroniclung inflammation.

[0336] The data presented in the Examples shows that SP-D is involved inthe regulation and clearance of apoptotic cells, and that rSPD(n/CRD)polypeptide, nucleic acid, or a fragment, homologue, variant orderivative thereof may be used to enhance the clearance of apoptoticcells such as apoptotic alveolar macrophages. Accordingly, rSPD(n/CRD)polypeptide, nucleic acid, or a fragment, homologue, variant orderivative thereof may be used to treat atherosclerosis, abnormalitiesof development, for example, abnormal post natal lung development inneonatal lung disease following premature birth, a disease associatedwith defective remodelling of inflamed tissues, cellular proliferationand cancer.

[0337] Atherosclerosis

[0338] In a preferred embodiment of the invention, the inflammatorydisease comprises atherosclerosis.

[0339] Atherosclerosis is a muli-factorial disease based on the actionof various risk factors that become effective on an appropriate geneticbackground. Atherosclerosis is characterised by the formation of foamcells (i.e. macrophages and smooth muscle cells that have taken upchemically modified eg. Oxidised low density lipoproteins (oxLDL). Wehave demonstrated the lung phenotype of SP-D knock-out mice, as shown inthe Examples, in particular that macrophages are highly abnormal andfoamy and there are disturbances of phospholipid turnover in the lung.It is known that SP-D is present in appreciable amounts in the serum,especially in certain disease states.

[0340] We therefore provide that disruption of lipid turnover in theserum confers an increased risk of atherosclerosis. Accordingly, rSP-D(N/CRD) may be used to treat atherosclerosis.

[0341] We have measured cholesterol levels in the serum of wild type andknock-out mice and show that cholesterol levels are higher in knock-outmice (See FIG. 17). High levels of cholesterol are known to confer anincreased risk of atherosclerosis. We therefore disclose thatsupplementation with recombinant SP-D, for example, rSP-D (N/CRD)intravenously, corrects this abnormality.

[0342] Furthermore, it has recently been shown that apoptosis plays amajor role in modulating the generation of atherosclerotic lesions invascular cells (Martinet W, Kockx M M. Apoptosis in atherosclerosis:focus on oxidized lipids and inflammation. Curr Opin Lipidol. 2001October; 12(5):535-41; Kockx M M, Herman A G. Apoptosis inatherosclerosis: beneficial or detrimental? Cardiovasc Res. 2000February;45(3):736-46). Apoptosis of macrophages could be beneficial forplaque stability provided apoptotic bodies are removed. Howeverapoptotic cells that are not scavenged in the plaque activate thrombinwhich could further induce intraplaque thrombosis. It can be concludedthat apoptosis in primary atherosclerosis is detrimental since it couldlead to plaque rupture and thrombosis. We show in the Examples thattreatment with recombinant SP-D reduces numbers of apoptotic macrophagesin the lungs of SP-D knock-out mice.

[0343] Therefore, a potential application of recombinant SP-Dadministered intravenously is the reduction of detrimental apoptoticcells (eg. Macrophages) in the vasculature, which may reduce the risk ofdevelopment of atherosclerosis. rSP-D (N/CRD) can therefore be used totreat or prevent any one or more of the symptoms of atherosclerosis.

[0344] Pharmaceutical Compositions

[0345] We note that rSPD(n/CRD) polypeptides may be produced in largeamounts at low cost in a bioactive form, allowing for the first time thedevelopment of rSP-D (N/CRD) containing surfactant formulations or ofco-administration of rSP-D (N/CRD) separately by aerosolisation,nebulisation, intranasal or intratracheal administration. Thepreparation is stable and maintains bioactivity after freeze-drying andresuspension, facilitating storage. Unlike current therapy withcorticosteroids which have undesirable systemic side effects, rSP-D(N/CRD) appears to have no ill effects in mice and is unlikely togenerate side-effects in humans.

[0346] While it is possible for the composition comprising therSPD(n/CRD) polypeptide or nucleic acid to be administered alone, it ispreferable to formulate the active ingredient as a pharmaceuticalformulation. We therefore also disclose pharmaceutical compositionscomprising rSPD(n/CRD) polypeptide or nucleic acid, or a fragment,homologue, variant or derivative thereof. Such pharmaceuticalcompositions are useful for delivery of rSPD(n/CRD) polypeptide, nucleicacid, fragment, homologue, variant or derivative thereof to anindividual for the treatment or alleviation of symptoms as described.

[0347] The composition may include the rSPD(n/CRD) polypeptide, nucleicacid, fragment, homologue, variant or derivative thereof, a structurallyrelated compound, or an acidic salt thereof. The pharmaceuticalformulations comprise an effective amount of rSPD(n/CRD) polypeptide,nucleic acid, fragment, homologue, variant or derivative thereof,together with one or more pharmaceutically-acceptable carriers. An“effective amount” of an rSPD(n/CRD) polypeptide, nucleic acid fragment,homologue, variant or derivative thereof is the amount sufficient toalleviate at least one symptom of a disease as described, for example,an inflammatory disease, preferably eczema, an inflammatory lungdisease, neonatal chronic lung disease, neonatal respiratory distresssyndrome (RDS), adult respiratory distress syndrome, chronic obstructiveairways disease (COPD), asthma, cystic fibrosis, pulmonary fibrosis,emphysema, interstitial inflammatory lung disease, sarcoidosis,pneumonia, chronic inflammatory lung disease, neonatal chronicinflammatory lung disease, an allergy, allergy is to house dust mite(Dermatophagoides app), preferably Dermatophagoides pteronyssinus orDermatophagoides farinae, or to fungi or fungal spores, preferablyAspergillus fumigatus, a seasonal respiratory allergy, allergicrhinitis, hayfever, nonallergic rhinitis, vasomotor rhinitis, irritantrhinitis, an allergy against grass pollens, tree pollens or animaldanders, an allergy associated with allergic asthma, a food allergy,microbial infection, including bacterial infection and viral infection,preferably a microbial infection of the lung.

[0348] The effective amount will vary depending upon the particulardisease or syndrome to be treated or alleviated, as well as otherfactors including the age and weight of the patient, how advanced thedisease etc state is, the general health of the patient, the severity ofthe symptoms, and whether the rSPD(n/CRD) polypeptide, nucleic acid,fragment, homologue, variant or derivative thereof is being administeredalone or in combination with other therapies.

[0349] Suitable pharmaceutically acceptable carriers are well known inthe art and vary with the desired form and mode of administration of thepharmaceutical formulation. For example, they can include diluents orexcipients such as fillers, binders, wetting agents, disintegrators,surface-active agents, lubricants and the like. Typically, The carrieris a solid, a liquid or a vaporizable carrier, or a combination thereof.Each carrier should be “acceptable” in the sense of being compatiblewith the other ingredients in the formulation and not injurious to thepatient. The carrier should be biologically acceptable without elicitingan adverse reaction (e.g. immune response) when administered to thehost.

[0350] The pharmaceutical compositions disclosed here include thosesuitable for topical and oral administration, with topical formulationsbeing preferred where the tissue affected is primarily the skin orepidermis (for example, psoriasis, eczema and other epidermal diseases).The topical formulations include those pharmaceutical forms in which thecomposition is applied externally by direct contact with the skinsurface to be treated. A conventional pharmaceutical form for topicalapplication includes a soak, an ointment, a cream, a lotion, a paste, agel, a stick, a spray, an aerosol, a bath oil, a solution and the like.Topical therapy is delivered by various vehicles, the choice of vehiclecan be important and generally is related to whether an acute or chronicdisease is to be treated. As an example, an acute skin proliferationdisease generally is treated with aqueous drying preparations, whereaschronic skin proliferation disease is treated with hydratingpreparations. Soaks are the easiest method of drying acute moisteruptions. Lotions (powder in water suspension) and solutions(medications dissolved in a solvent) are ideal for hairy andintertriginous areas. Ointments or water-in-oil emulsions, are the mosteffective hydrating agents, appropriate for dry scaly eruptions, but aregreasy and depending upon the site of the lesion sometimes undesirable.As appropriate, they can be applied in combination with a bandage,particularly when it is desirable to increase penetration of the agentcomposition into a lesion. Creams or oil-in-water emulsions and gels areabsorbable and are the most cosmetically acceptable to the patient.(Guzzo et al, in Goodman & Gilman's Pharmacological Basis ofTherapeutics, 9th Ed., p. 1593-15950 (1996)). Cream formulationsgenerally include components such as petroleum, lanolin, polyethyleneglycols, mineral oil, glycerin, isopropyl palmitate, glyceryl stearate,cetearyl alcohol, tocopheryl acetate, isopropyl myristate, lanolinalcohol, simethicone, carbomen, methylchlorisothiazolinone,methylisothiazolinone, cyclomethicone and hydroxypropyl methylcellulose,as well as mixtures thereof.

[0351] Other formulations for topical application include shampoos,soaps, shake lotions, and the like, particularly those formulated toleave a residue on the underlying skin, such as the scalp (Arndt et al,in Dermatology In General Medicine 2:2838 (1993)).

[0352] In general, the concentration of the rSPD(n/CRD) polypeptide,nucleic acid, fragment, homologue, variant or derivative thereofcomposition in the topical formulation is in an amount of about 0.5 to50% by weight of the composition, preferably about 1 to 30%, morepreferably about 2-20%, and most preferably about 5-10%. Theconcentration used can be in the upper portion of the range initially,as treatment continues, the concentration can be lowered or theapplication of the formulation may be less frequent. Topicalapplications are often applied twice daily. However; once-dailyapplication of a larger dose or more frequent applications of a smallerdose may be effective. The stratum corneum may act as a reservoir andallow gradual penetration of a drug into the viable skin layers over aprolonged period of time.

[0353] In a topical application, a sufficient amount of activeingredient must penetrate a patient's skin in order to obtain a desiredpharmacological effect. It is generally understood that the absorptionof drug into the skin is a function of the nature of the drug, thebehaviour of the vehicle, and the skin. Three major variables accountfor differences in the rate of absorption or flux of different topicaldrugs or the same drug in different vehicles; the concentration of drugin the vehicle, the partition coefficient of drug between the stratumcorneum and the vehicle and the diffusion coefficient of drug in thestratum corneum. To be effective for treatment, a drug must cross thestratum corneum which is responsible for the barrier function of theskin. In general, a topical formulation which exerts a high in vitroskin penetration is effective in vivo. Ostrenga et al (J. Pharm. Sci.,60:1175-1179 (1971) demonstrated that in vivo efficacy of topicallyapplied steroids was proportional to the steroid penetration rate intodermatomed human skin in vitro.

[0354] A skin penetration enhancer which is dermatologically acceptableand compatible with the agent can be incorporated into the formulationto increase the penetration of the active compound(s) from the skinsurface into epidermal keratinocytes. A skin enhancer which increasesthe absorption of the active compound(s) into the skin reduces theamount of agent needed for an effective treatment and provides for alonger lasting effect of the formulation. Skin penetration enhancers arewell known in the art. For example, dimethyl sulfoxide (U.S. Pat. No.3,711,602); oleic acid, 1,2-butanediol surfactant (Cooper, J. Pharm.Sci., 73:1153-1156 (1984)); a combination of ethanol and oleic acid oroleyl alcohol (EP 267,617), 2-ethyl-1,3-hexanediol (WO 87/03490); decylmethyl sulphoxide and Azone.RTM. (Hadgraft, Eur. J. Drug. Metab.Pharmacokinet, 21:165-173 (1996)); alcohols, sulphoxides, fatty acids,esters, Azone.RTM., pyrrolidones, urea and polyoles (Kalbitz et al,Pharmazie, 51:619-637 (1996));

[0355] Terpenes such as 1,8-cineole, menthone, limonene and nerolidol(Yamane, J. Pharmacy & Pharmocology, 47:978-989 (1995)); Azone.RTM. andTranscutol (Harrison et al, Pharmaceutical Res. 13:542-546 (1996)); andoleic acid, polyethylene glycol and propylene glycol (Singh et al,Pharmazie, 51:741-744 (1996)) are known to improve skin penetration ofan active ingredient.

[0356] Levels of penetration of an agent or composition can bedetermined by techniques known to those of skill in the art. Forexample, radiolabeling of the active compound, followed by measurementof the amount of radiolabeled compound absorbed by the skin enables oneof skill in the art to determine levels of the composition absorbedusing any of several methods of determining skin penetration of the testcompound. Publications relating to skin penetration studies includeReinfenrath, W G and G S Hawkins. The Weaning Yorkshire Pig as an AnimalModel for Measuring Percutaneous Penetration. In:Swine in BiomedicalResearch (M. E. Tumbleson, Ed.) Plenum, N.Y., 1986, and Hawkins, G. S.Methodology for the Execution of In Vitro Skin PenetrationDeterminations. In: Methods for Skin Absorption, B W Kemppainen and W GReifenrath, Eds., CRC Press, Boca Raton, 1990, pp.67-80; and W. G.Reifenrath, Cosmetics & Toiletries, 110:3-9 (1995).

[0357] For some applications, it is preferable to administer a longacting form of agent or composition using formulations known in thearts, such as polymers. The agent can be incorporated into a dermalpatch (Junginger, H. E., in Acta Pharmaceutica Nordica 4:117 (1992);Thacharodi et al, in Biomaterials 16:145-148 (1995); Niedner R., inHautarzt 39:761-766 (1988)) or a bandage according to methods known inthe arts, to increase the efficiency of delivery of the drug to theareas to be treated.

[0358] Optionally, the topical formulations of this invention can haveadditional excipients for example; preservatives such as methylparaben,benzyl alcohol, sorbic acid or quaternary ammonium compound; stabilizerssuch as EDTA, antioxidants such as butylated hydroxytoluene or butylatedhydroxanisole, and buffers such as citrate and phosphate.

[0359] The pharmaceutical composition can be administered in an oralformulation in the form of tablets, capsules or solutions. An effectiveamount of the oral formulation is administered to patients 1 to 3 timesdaily until the symptoms of the disease alleviated. The effective amountof agent depends on the age, weight and condition of a patient. Ingeneral, the daily oral dose of agent is less than 1200 mg, and morethan 100 mg. The preferred daily oral dose is about 300-600 mg. Oralformulations are conveniently presented in a unit dosage form and may beprepared by any method known in the art of pharmacy. The composition maybe formulated together with a suitable pharmaceutically acceptablecarrier into any desired dosage form. Typical unit dosage forms includetablets, pills, powders, solutions, suspensions, emulsions, granules,capsules, suppositories. In general, the formulations are prepared byuniformly and intimately bringing into association the agent compositionwith liquid carriers or finely divided solid carriers or both, and asnecessary, shaping the product. The active ingredient can beincorporated into a variety of basic materials in the form of a liquid,powder, tablets or capsules to give an effective amount of activeingredient to treat the disease.

[0360] Other therapeutic agents suitable for use herein are anycompatible drugs that are effective for the intended purpose, or drugsthat are complementary to the agent formulation. The formulationutilized in a combination therapy may be administered simultaneously, orsequentially with other treatment, such that a combined effect isachieved.

[0361] Surfactant Therapy Compositions

[0362] The development of rSP-D (N/CRD) containing surfactant therapiesmay be applied to the treatment of neonatal respiratory distresssyndrome, in place of current formulations or in conjunction withcurrent formulations.

[0363] Current surfactant therapy for newborn respiratory distresssyndrome includes the following:

[0364] Beractant (Survanta)

[0365] Beractant (Survanta) is a semisynthetic bovine lung extractcontaining phospholipids, fatty acids, and surfactant-associatedproteins B (7 mcg/mL) and C (203 mcg/mL). Beractant is delivered at aPediatric Dose of 100 mg (ie, 4 mL)/kg divided in 4 aliquotsadministered at least 6 h apart (Intratracheal).

[0366] Calfactant (Infasurf)

[0367] Calfactant (Infasurf) is a natural calf lung extract containingphospholipids, fatty acids, and surfactant-associated proteins B. it ismanufactured by Ross, and is delivered at (260 mcg/mL) and C (390mcg/mL).

[0368] SURVANTA® (beractant) Intratracheal Suspension

[0369] SURVANTA® (beractant) is a sterile, non-pyrogenic pulmonarysurfactant intended for intratracheal use. It is a natural bovine lungextract containing phospholipids, neutral lipids, fatty acids, andsurfactant-associated proteins to which colfosceril palmitate(dipalmitoylphosphatidylcholine), palmitic acid, and tripalmitin areadded to standardize the composition and to mimic surface-tensionlowering properties of natural lung surfactant. The resultingcomposition provides 25 mg/mL phospholipids (including 11.0-15.5 mg/mLdisaturated phosphatidylcholine), 0.5-1.75 mg/mL triglycerides, 1.4-3.5mg/mL free fatty acids, and less than 1.0 mg/mL protein. It is suspendedin 0.9% sodium chloride solution, and heat-sterilized. SURVANTA containsno preservatives. Its protein content consists of two hydrophobic, lowmolecular weight, surfactant-associated proteins commonly known as SP-Band SP-C. It does not contain the hydrophilic, large molecular weightsurfactant-associated protein known as SP-A.

[0370] INFRASURF Intratracheal Suspension (Forest)

[0371] Infasurf® (calfactant) Intratracheal Suspension is a sterile,non-pyrogenic lung surfactant intended for intratracheal instillationonly. It is an extract of natural surfactant from calf lungs whichincludes phospholipids, neutral lipids, and hydrophobicsurfactant-associated proteins B and C (SP-B and SP-C). It contains nopreservatives.

[0372] Infasurf is an off-white suspension of calfactant in 0.9% aqueoussodium chloride solution. It has a pH of 5.0-6.0. Each milliliter ofInfasurf contains 35 mg total phospholipids (including 26 mgphosphatidylcholine of which 16 mg is disaturated phosphatidylcholine)and 0.65 mg proteins including 0.26 mg of SP-B.

[0373] EXOSURF Neonatal for Intratracheal Suspension (Glaxo Wellcome)

[0374] EXOSURF NEONATAL (colfosceril palmitate, cetyl alcohol,tyloxapol) for Intratracheal Suspension is a protein-free synthetic lungsurfactant stored under vacuum as a sterile lyophilized powder. EXOSURFNEONATAL is reconstituted with preservative-free Sterile Water forInjection prior to administration by intratracheal instillation. Each10-mL vial contains 108 mg colfosceril palmitate, commonly known asdipalmitoylphosphatidylcholine (DPPC), 12 mg cetyl alcohol, 8 mgtyloxapol, and 47 mg sodium chloride. Sodium hydroxide or hydrochloricacid may have been added to adjust pH. When reconstituted with 8 mLSterile Water for Injection, the EXOSURF NEONATAL suspension contains13.5 mg/mL colfosceril palmitate, 1.5 mg/mL cetyl alcohol, and 1 mg/mLtyloxapol in 0.1 N NaCl. The suspension appears milky white with a pH of5 to 7 and an osmolality of 185 mOsm/kg.

[0375] The chemical names of the compounds of EXOSURF NEONATAL are asfollows: colfosceril palmitate:(R)-4-hydroxy-N,N,N-trimethyl-10-oxo-7-[(1-oxohexadecyl)oxy]-3,5,9-trioxa-4-phosphapentacosan-1-aminiumhydroxide inner salt, 4-oxide; cetyl alcohol: (1-hexadecanol); andtyloxapol: 4-(1,1,3,3-tetramethylbutyl)phenol polymer with formaldehydeand oxirane.

[0376] In EXOSURF NEONATAL, which is protein free, cetyl alcohol acts asthe spreading agent for the DPPC on the air-fluid interface. Tyloxapol,a polymeric long-chain repeating alcohol, is a nonionic surfactant whichacts to disperse both DPPC and cetyl alcohol. Sodium chloride is addedto adjust osmolality.

[0377] CUROSURF®

[0378] Brand Name: Curosurf Active Ingredient: poractant alfaStrength(s): 120 mg of phospholipid/1.5 mL or 240 mgphospholipid/3 mLvials Dosage Form(s): Intrathracheal suspension Company Name: Dey, LPAvailability: Prescription only, for professional use only *DateApproved by the FDA: Nov. 18, 1999.

[0379] Accordingly, we disclose the use of compositions including any ofthe ingredients of the therapies disclosed above, preferably an activeingredient, in conjunction with rSPD(n/CRD) polypeptide, nucleic acid,or a fragment, homologue, variant or derivative thereof, as a surfactanttherapy. Thus, we disclose a composition comprising a lung extracttogether with an rSPD(n/CRD) polypeptide, nucleic acid, or a fragment,homologue, variant or derivative thereof. The lung extract may comprisea bovine, preferably, calf lung extract together with phospholipids(such as phosphatidylcholine and disaturated phosphatidylcholine) and/orfatty acids. The composition may further include a surfactant-associatedprotein, for example a surfactant-associated protein B or asurfactant-associated protein C. Other components such as cetyl alcohol,long-chain repeating alcohols, tyloxapol, colfosceril palmitate(dipalmitoylphosphatidylcholine), palmitic acid, and tripalmitin,poractant alfa and sodium chloride may also be added.

[0380] In particular, we disclose compositions corresponding to each ofBeractant (Survanta); Calfactant (Infasurf); SURVANTA® (beractant) ;INFRASURF; EXOSURF Neonatal; and CUROSURF; in which each compositionfurther comprises a therapeutically effective amount of an rSPD(n/CRD)polypeptide, nucleic acid, or a fragment, homologue, variant orderivative thereof.

[0381] Further Aspects

[0382] Further aspects include the following:

[0383] A method of enhancing MCP-1 levels or MIP1 α levels, or both, inan individual, the method comprising administering to the individual anrSPD(n/CRD) polypeptide, fragment, homologue, variant or derivativethereof

[0384] A method of enhancing cytokine levels, preferably IFNγ levels, inan individual, the method comprising administering to the individual anrSPD(n/CRD) polypeptide, fragment, homologue, variant or derivativethereof.

[0385] A method of up-regulating a component of the cell-mediated immunesystem, or up-regulating the activity of natural killer cells, or both,in an individual by administration of an rSPD(n/CRD) polypeptide,fragment, homologue, variant or derivative thereof is also disclosed.

EXAMPLES EXAMPLES Example 1 Materials and Methods

[0386] Recombinant Surfactant Protein D Fragment (rSP-D (N/CRD))

[0387] The recombinant Surfactant Protein D Fragment (rSP-D (N/CRD))used in these experiments is a truncated form of human SP-D. Weexpressed the fragment in E.coli from a plasmid containing cDNA for thefragment of human SP-D (the construct) inserted into a pET vector andtransformed into E.coli for expression using standard fermentationprocedures.

[0388] The construct is composed of a short stretch of 8 N-terminalGly-XaaYaa triplets (shown in italics) with a substitution of serine forproline at position 2 followed by the 28 residue neck region (shown inbold) and the 125 residue Carbohydrate Recognition Domain (CRD) of humanSP-D. GSPGLKGDKGIPGDKGAKGESGLP DVASLRQQVEALQGQVQHLQAAFSQYKKVELFPNGQSVGEKIFKTAGFVKPFTEAQLLCTQAGGQLASPRSAAENAALQQLVVAKNEAAFLSMTDSKTEGKFTYPTGESLVYSNWAPGEPNDDGGSEDCVEIFTNGKWNDRACGEKRLVVCEF

[0389] A schematic diagram showing the structure of rSP-D (N/CRD) isshown in FIG. 1A. (A): Surfactant Protein D dodecamer; (B): SP-D trimer;(C): Ribbon diagram of the overall structure of the head-neck region ofSP-D.

[0390] A nucleic acid sequence encoding rSPD(n/CRD) polypeptide has thesequence shown in SEQ ID NO: 2.

[0391] Generation of Expression Construct of rSP-D (N/CRD) in pET vectorpET21d

[0392] A sequence encoding the human SP-D (neck/CRD) and including ashort stretch encoding 8 N-terminal Gly-XaaYaa triplets was excised froma previously generated human SP-D clone pBCSK1 (Kishore, U., et al., Thealpha-helical neck region of human lung surfactant protein D isessential for the binding of the carbohydrate recognition domains tolipopolysaccharides and phospholipids. Biochem J, 1996. 318(Pt 2): p.505-11.) by digestion with Xba 1 and Hind-3. The resulting fragment isthen purified by agarose electrophoresis and ligated into a pET-21dvector (Novagen) which is digested with Nhe1 and Hind 3. The pET-21dvector is available from Novegen under catalogue number 69743-3. Itssequence is known in the art, and is described in for example,literature from Novagen (e.g., as found at http://www.novagen.com/).

[0393] Preparation of rSP-D (N/CRD)

[0394] The new construct so generated was then used to transformcompetent cells (DH5α) Plasmid DNA comprising this construct forrSP-D(neck/CRD) is purified after growth of cells from transformedcolony by standard methods.

[0395] Plasmid containing cDNA for rSPD(n/CRD) (neck/CRD) in a pETvector is transformed into BL21(XDE3) pLysS and a single colony selectedand diluted into I nil LB supplemented with 100 μg/ml ampicillin and 25μg/ml chloramphenicol (LB) This is further diluted 1000 fold and 100 μlspread onto an agar plate and incubated overnight. The resulting lawn ofcolonies is collected by scraping with a flat bladed spatula andresuspended in 50 ml LB⁺. An aliquot of 5 ml of this is used toinoculate 2 L flasks containing 500 ml LB. This procedure is found to besuperior to the more common practice of growing up an overnight culturein growth medium. The growth of colonies on a stationary phase reducesthe contact between cells and reduces the proportion of mutants in theinoculum which do not carry the expression plasmid.

[0396] It is also found to be important not to exceed 500 ml LB/2L flaskin order to achieve optimum expression levels.

[0397] Cultures are grown to A₆₀₀ of 0.6-0.8 followed by induction with0.4 mM IPTG for 2-3 hr.

[0398] Purification of Recombinant Surfactant Protein D Fragment rSP-D(N/CRD)

[0399] Cells are harvested by centrifugation and lysed in 20 mMTris-HCl, 150 mM NaCl, 5 mM EDTA, 0.1% v/v Triton X-100, 0.1 mM PMSF, pH7.5) and sonicated for 3 minutes. The rSPD(n/CRD) is expressed ininsoluble inclusion bodies and is harvested by centrifugation at10000×g. This step is repeated three times and the purified inclusionbody pellet, containing the rSPD(n/CRD), was solubilized in 100 ml of 8M Urea, 100 mM 2-mercaptoethanol, pH 7.5 and clarified by high speedcentrifugation.

[0400] The urea solution is diluted into I L 20 mM TrisHCl, pH 7.5 and50 ml of Q-Sepherose anion exchange resin (Pharmacia) added and mixed at20° C. for 30 mm. The adsorbed resin was is into an FPLC column, washedextensively with 20 mM Tris-HCl, 150 mM NaCl, pH 7.5, and therSPD(n/CRD) is eluted with 50 mM Tris-HCl, 500 mM NaCl, pH 7.5.

[0401] The protein from the peak fractions is precipitated by a 10:1dilution into acetone at 4° C. for 30 mm. The precipitate is harvestedby high speed centrifugation and dissolved in 100 ml of 8 M Urea anddialysed against 10 L of 20 mM Tris-HCl, 150 mM NaCl, 2 mM CaCl₂, pH7.4, overnight at 4° C.

[0402] The dialysate is then mixed with 20 nil of maltose-agarose forseveral hours to over-night in 20 mM Tris-HCl, 10 mM CaCl₂, pH 7.4. Thislong incubation step probably facilitates the final stages of re-foldinginto active protein.

[0403] The maltose-agarose is packed into an FPLC column and washed withthe same buffer containing 1M NaCl to remove non-specific proteins. Thehigh salt wash elutes approximately 25-50% of the total 01)280 absorbingmaterial bound to the column. SDS-PAGE shows traces of rSPD(n/CRD)(approximately 10%), which is presumably low affinity rSPD(n/CRD) thatmay not be fully folded into the correct 3D structure. As thesepreparations of rSPD(n/CRD) are to be used in structural studies and inexperiments in mice, it is considered advisable not to include thisfraction. In addition the total 0D₂₈₀ absorbing material in the highsalt is far greater than could be attributed to this rSPD(n/CRD) and isprobably due to bacterial DNA and other substances that are deemedundesirable in a preparation for administering to animals.

[0404] The bound rSPD(n/CRD) was then eluted with 20 mM Tris-HCl, 150 mMNaCl, 0.02% (w/v) sodium azide pH 7.4, containing 5 mM EDTA to removethe Ca required for rSPD(n/CRD) CRD mediated binding.

[0405] The peak fractions are concentrated using an Amicon stirred cellwith a 10 000 MWCO membrane to 5 ml and loaded onto a 100 ml Superose 12gel filtration column (Pharmacia) in a running buffer of 20 mM Tris-HCl,150 mM NaCl, 5 mM EDTA, 0.02% (w/v) sodium azide pH 7.4. The rSPD(n/CRD)eluted as a single peak corresponding to 60 kDa molecular weight.

[0406] This step allows the final dissociation of any impurities thatmight have co-purified to this stage and in the presence of EDTA anycarbohydrates will dissociate from the rSPD(n/CRD) and would be expectedto elute in the total inclusion volume of the column (100 ml) since theyshould have a lower molecular weight.

[0407] This step also allows an assessment of the homogeneity of theprotein preparation by the symmetry of the protein peak and absence ofshoulders. Fractions outside the main peak are discarded. The pooledfractions are collected and concentrated to 2 mg/ml before proceeding tothe next step. The recovery of rSPD(n/CRD) after gel filtration istypically 80% of the total OD₂₈₀ absorbing material loaded. Thisindicates that at least 20% of this is not due to rSPD(n/CRD) andreinforces the need for including gel filtration as a final step.

[0408] Endotoxin levels are reduced by passing the purified rSPD(n/CRD)in 20 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 0.02% (w/v) sodium azide pH7.4 through a 10 ml Polymixin B column Detoxi-Gel, Pierce).

[0409] Modification of rSP-D by FITC Labelling

[0410] To assess in vitro binding to macrophages isolated by BAL ofmice, rSP-D is labelled with the amine reactive probe FITC (Sigma)according to the manufacturer's instructions. Briefly, 50 μg of FITC isincubated with 1 mg rSP-D in 200 ml of 0.1M sodium bicarbonate buffer pH9.0 at room temperature for 2 hours.

[0411] The labelled protein is then separated from free FITC using aG-25 column. FITC-rSP-D (20 μg/ml) is incubated with freshly isolatedalveolar macrophages from wild-type and SP-D knock-out mice in bindingbuffer (10 mM Hepes, 140 mM NaCl, 2.5 mM CaCl₂) and assessed forco-labelling with propidium iodide or PB-labeled annexin V as outlinedbelow by flow cytometry on a Consort 32 FACs System.

[0412] Preparation of Aspergillus fumigatus Antigen (Afu lwcf)

[0413]Aspergillus fumigatus (Afu) is grown in a synthetic medium (M199,Sigma Chemicals) as a stationary culture for 1 week at 37° C. Arrunda,et al, (Arruda, L. K., B. J. Mann, and M. D. Chapman. 1992. Selectiveexpression of a major allergen and cytotoxin, Asp f I, in Aspergillusfumigatus. Implications for the immunopathogenesis ofAspergillus-related diseases. J Immunol. 149:3354-9.) demonstrates thatthe expression of Asp f1, a major allergen, is maximal after 1 week andtends to diminish during longer incubation periods.

[0414] The 1 week culture is killed by adding 0.1% Thimerosal for 12hours. The culture is filtered through glass wool and finally through a0.45 μm membrane to remove all particulates and possible spores and thendialysed with 3 buffer changes against water. The dialysate islyopholised to give a brown powder.

[0415] There is a major band at 18 kDa, which corresponds to Asp f 1. Aband corresponding to Asp f2 (37 kDa) is also evident. The 18 kDa bandis N-terminal sequenced giving the sequence ATWTCINQQLNP, correspondingto the N-terminal sequence for Asp f 1.

[0416] It is also demonstrated by ELISA that the 1-week culture filtrate(1wcf) is recognised by human serum from Afu-allergic patients obtainedfrom the National Institute of Biological Standards and Control.

[0417] Transgenic Strains

[0418] We have previously reported the generation of gene-targeted SP-Ddeficient mice (Botas, C., Poulain, F., Akiyama, J., Brown, C., Allen,L., Goerke, J., Clements, J., Carlson, E., Gillespie, A. M., Epstein, C.& Hawgood, S. (1998) Proc Natl Acad Sci USA 95, 11869-74.).

[0419] SP-D deficient mice backcrossed 10 generations into a C57B16background are fed ad libitum and housed in isolators in a pathogen freeenvironment in the Biomedical Services Unit, Oxford University.Pathogen-free C57B16 wild-type mice for control experiments are obtainedfrom Harlan-OLAC, Shaw's farm, Bicester, Oxfordshire. All experimentalprotocols are approved by appropriate U.K. Home Office licensingauthorities and by the University of Oxford Ethical Committee.

[0420] Administration of rSP-D, rSP-A, and Bovine Serum Albumin (BSA)

[0421] 6-week old SP-D deficient mice underwent multiple intranasaladministrations of protein or PBS alone over a period of 3-6 weeks usingone of three protocols.

[0422] For each protein administration mice are lightly anaesthetisedwith isofluorane before rSP-D or control protein (30 μg, 25 μg, or 10 μgof rSP-D, rSP-A or BSA in 50 μl of PBS) is applied to the nares using asterile micropipette. Mice are held upright after each dose until all ofthe fluid is inhaled. In the first treatment protocol twelve micereceived 30 μg doses of rSP-D every three days from age six weeks. Sixmice are sacrificed after three weeks for assay of alveolar macrophagenumber and alveolar phospholipid content. The remaining six mice in thetreatment group completed six weeks of treatment before sacrifice andassay. In the second treatment protocol, mice are, treated from age 12weeks with 10 μg doses of rSP-D, rSP-A, BSA five times per week forthree weeks before sacrifice and assay. Additional controls areuntreated mice and mice treated with PBS. To assess the effect oftreatment from age four weeks, before a significant increase in alveolarmacrophage numbers occurs, six mice are treated in a third protocol fortwo weeks with 30 μg doses of rSP-D. These mice are sacrificed at agesix weeks for assay of phospholipids and alveolar macrophage numbers,and compared to untreated age matched controls.

[0423] Bronchoalveolar Lavage (BAL)

[0424] For isolation of alveolar macrophages (for example for anapoptosis assay), 4-6 mice in each treatment group are sacrificed byasphyxiation with carbon dioxide and undergo bronchoalveolar lavage(BAL) with sterile RPMI.

[0425] A sterile cannula is inserted into the trachea and tied inposition with thread. Using a 2 ml syringe, the lungs are lavaged with 1ml volumes of lavage buffer, four times to yield a total lavage volumeof approximately 3 ml. BAL fluid is then centrifuged at 250 g for 5minutes at room temperature to pellet alveolar macrophages. Cells arewashed by resuspension in 1 ml of PBS and pelleted again bycentrifugation in a benchtop centrifuge at 1000 rpm for 2 minutes. Cellsare then resuspended and incubated with FITC-labelled Annexin V andPropidium iodide for 10 minutes. Samples are then analysed for stainingby FACS flow cytometry.

[0426] Cytospin Preparations of Alveolar Macrophages

[0427] Alveolar macrophages isolated by bronchoalveolar laveage usingPBS with 0.25 mM EDTA and centrifugation at 250 g are resuspended in 1ml of PBS. Aliquots are taken for total cell counting by hemocytometerafter staining with malachite green or crystal violet and preparation ofcytospin slides using standard procedures. Differential cell counts oncytospin preparations after staining with Diff-Quik (ScientificProducts, McGaw Park, Ill.) confirm that 98% of the cells isolated inthis way are alveolar macrophages.

[0428] In Vitro Labelling of Mouse Alveolar Macrophages

[0429] Alveolar macrophages isolated from BAL of wild-type and knock-outmice are labelled in vitro with fluorescent green and orange celltracker dyes. Cell Tracker™ Orange consisting of CMTMR(5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine mixedisomers and Cell Tracker™.

[0430] Green (Molecular probes, Eugene, Oreg.) consisting of CMFDA(5-chloromethylfluorescein diacetate) contain a mildly thiol reactivechloreomethyl group. Once inside the cell, the chloromethyl group reactswith intracellular thiols, transforming the probe into a cell impermeantfluorescent dye ether adduct. Excess unconjugated reagent passeivelydiffuses to the extracellular medium. Cell Tracker Orange does not needesterase activity to be fluorescent, whereas cell tracker green iscolourless until cleaved by intracellular esterases.

[0431] Bronchoalveolar Lavage Fluid Total Phospholipid and ProteinMeasurements

[0432] At the time points indicated in individual experiments, 4-6 micein each treatment group are sacrificed by asphyxiation with carbondioxide and underwent bronchoalveolar lavage (BAL) with sterile PBScontaining 0.25 mM EDTA. The lungs are lavaged with Imi four times toyield a total lavage volume of approximately 3 ml. The BAL fluid iscentrifuged at 250 g for 5 minutes at room temperature. Total proteinconcentration in the cell free supernatant is determined usingbicinchoninic acid as a substrate. Cell free BAL fluid is extracted intochloroform methanol and the total phospholipid derived from thephosphorous concentration.

[0433] rSP-D Levels in Mouse BAL Fluid

[0434] Serial dilutions of cell free BALF from rSP-D treated mice areanalysed for rSP-D content by standard sandwich ELISA methodology usingbiotinylated and non-biotinylated, monospecific, polyclonal antibodiesraised against recombinant human SP-D. This antibody showed no crossreactivity with mouse SP-A or mouse SP-D. Standard curves usingrecombinant human SP-D are used to calculate the absolute amounts ofalveolar rSP-D recovered at specific time points alter administration.

[0435] Flow Cytometry and Detection of Apoptotic and Necrotic Cells

[0436] Cells isolated from BAL of SP-D deficient mice with RPMI areresuspended in PBS and analysed on a Consort 32 FACs System (BectonDickinson Immunocytometry Systems, San Jose, Calif.) to characterise themacrophage population by size and granularity. Cell preparations withobvious blood staining are discarded. Apoptotic cells and necrotic cellsare detected by annexin V and propidium iodide (P1) staining using anAnnexin-V-FLUOS staining kit (Roche Diagnostics, Mannheim, Germany).Apoptotic and necrotic cells expose phosphatidylserine (PS), which isnormally present on the inner cell membrane leaflet to the outerleaflet, allowing annexin V to bind to PS at the cell surface (Vermes,I., Haanen, C., Steffens-Nakken, H. & Reutelingsperger, C. (1995) JImmunol Methods 184, 39-51.). Cell aliquots are stained withfluorescent-labelled annexin V and counterstained with PI to detectprimary or secondary necrotic cells. In co-labelling experiments,binding of FITC-labelled rSP-D to cells staining with PI or PE-labelledannexin V (Pharmingen, San Diego, Calif.) are also assessed aftercompensation for overlap of fluorescent signals.

[0437] Data Analysis

[0438] Results are given as means±standard errors of the mean (x±SE).Comparisons between groups of animals at individual time points are madewith two-tailed t-tests assuming unequal variance. Significance isaccepted at P<0.05.

Example 2 Modulation of Allergic Hypersentivity in a Murine Model ofAllergic Hypersensitivity to Aspergillus fumigatus

[0439] The truncated form of human SP-D composed of timers of the neckand CRD domains, expressed and purified in E.coli and referred to asrSPD(n/CRD), is functionally active as a modulator of allergichypersensitivity reactions in mice. In this present study, modulation ofeosinophilia, serum IgE and IgG1, has been reproduced in C57BU6, thusdemonstrating that the effects are not peculiar to BALB/c mice.

[0440] The parameters measured in the present study are serum IgE andIgG1 and peripheral blood eosinophilia which are all significantlyelevated in the mouse model of allergy to Aspergillus fumigatusallergens and all significantly reduced by intranasal treatment withrSPD(n/CRD).

[0441] Without seeming to be bound by any particular theory, it isproposed that rSPD(n/CRD) promotes a shift in the populations of Tlymphocytes from Th2 to Th1. This culminates in the observed reductionin serum IgE and eosinophilia, which are major components in allergy.

[0442] Preparation of rSPD(n/CRD)

[0443] The cDNA for the neck/CRD, including a short region of thecollagen stalk (8 Gly-X-Y) and representing residues 179-355 of themature protein sequence is cloned from human lung library DNA andinserted into a pET-21d vector (Novagen, Nottingham). The plasmid istransformed into BL21(λDE3) pLysS and a single colony selected andre-plated to give 100-400 colonies/plate. These are scraped and used toinoculate shake-flasks containing 500 ml LB medium supplemented with 100μg/ml ampicillin and 25 μg/ml chloramphenicol and grown to an OD600 of0.6-0.8 followed by induction with 0.4 mM IPTG for 2-3 hr. Cells arecollected by centrifugation and lysed in 20 mM Tris-HCl, 150 mM NaCl, 5mM EDTA, 0.1% v/v Triton X-100, 0.1 mM PMSF, pH 7.5) and sonicated for 3minutes. The rSPD(n/CRD) is expressed in insoluble inclusion bodies andis collected by centrifugation and washed 4 times at 10000×g. The pelletis solubilized in 100 ml of 8 M Urea, 100 mM 2-mercaptoethanol, pH 7.5and clarified by centrifugation and refolded by overnight dialysisagainst 10L of 20 mM Tns-HCl, 150 mM NaCl, 5 mM CaCl₂ (TCB). RefoldedrSPD(n/CRD) is separated from denatured rSPD(n/CRD) by absorption ontomaltose-agarose (Sigma-Aldrich, Poole, UK) and eluted with 20 mMTris-HCl, 150 mM NaCl, containing 5 mM EDTA after first washing thecolumn with TCB containing 1M NaCl to remove impurities. Finalpurification is by gel filtration column (Superose 12,AmershamPharmacia, UK) ) in a running buffer of 20 mM Tris-HCl, 150 mM NaCl, 5mM EDTA, 0.02% (w/v) sodium azide pH 7.4 (TSE). The rSPD(n/CRD) elutedas a single peak corresponding to 60 kDa molecular weight. Endotoxinlevels are reduced by passing the purified rSPD(n/CRD) in through a 10ml Polymixin B column (Detoxi-Gel, Pierce & Warriner, UK) and onlypreparations containing less than 5 pg/μg rSPD(n/CRD) are used.

[0444]Aspergillus fumigatus Antigen (Afu lwcf)

[0445]Aspergillus fumigatus (Mu) is grown in a synthetic medium (M199,Sigma Chemicals) as a stationary culture for 1 week at 37° C. Arruda, etal, [Arruda L K, Mann B J, Chapman M D. Selective expression of a majorallergen and cytotoxin, Asp f I, in Aspergillus fumigatus. Implicationsfor the immunopathogenesis of Aspergillus-related diseases. J Immunol1992; 149:3354-9] demonstrated that the expression of Asp f 1, a majorallergen, is maximal after 1 week and tends to diminish during longerincubation periods. The 1 week culture is killed by adding 0.1% (w/v)Thimerosal for 12 hours at 4° C. The culture is filtered through glasswool and finally through a 0.45 μm membrane to remove all particulatesand spores and then dialysed with 3 buffer changes against water. Thedialysate is lyopholised to give a brown powder. SDS PAGE (FIG. 1B) oflwcf revealed a major band at 18 kDa, which corresponds to Asp f1. Aband corresponding to Asp f 2 (37 kDa) is also evident. The 18 kDa bandis N-terminal sequenced giving the sequence ATWTCINQQLNP, correspondingto the N-terminal sequence for Asp f 1. It is also demonstrated by ELISAthat the 1-week culture filtrate (lwcf) is recognised by human serumfrom Afu-allergic patients obtained from the National Institute ofBiological Standards and Control.

[0446] Sensitisation

[0447] In this study, 6 week old female C57BL/6 mice are sensitised byintra-peritoneal (i.p.) injections of 200 μg Afu lwcf mixed withalum(1:4 v/v) in 100 μg PBS given once a week for 4 weeks.

[0448] Allergen Challenge and Treatment

[0449] Sensitised mice are challenged with 50 μl containing 10 μg of Afulwcf given intranasally (i.n.). This is followed by treatment with PBSor 10 μg rSPD(n/CRD) in 50 μl PBS given i.n. Challenge and treatment areperformed on a daily basis as described in the results. In someexperiments, a control protein of full length recombinant human SP-A(kindly provided by Byk Gulden Pharmaceuticals) is used at aconcentration of 10 μg/50 μl. In a separate experiment the fate ofrSPD(n/CRD) is monitored by obtaining BAL from different mice at varioustimes after intranasal application rSPD(n/CRD) in 50 μl PBS and assayingfor rSPD(n/CRD) using a polyclonal antibody raised against rSPD(n/CBD)that does not recognise mouse SP-D or SP-A. These results show that atleast 50% of rSPD(n/CRD) could be accounted for in the BAL taken 30minutes after administration and none could be measured after 24 hrs.

[0450] Peripheral Blood Eosinophils

[0451] Blood is collected from the tail vein of the mice (n=4-8/group)for estimation of eosinophils. Total leukocyte count is measured with anautomatic cell counter and the proportion of eosinophils is determinedby differential counting of May-Grunwald-Giemsa stained blood smears.Results are expressed as 10⁶ cells/ml.

[0452] Serum IgE and Afu-Specfic IgGl

[0453] Total serum IgE is measured by sandwhich ELISA (BD PharMingen,Cowley, UK) in blood serially diluted from a maximum dilution of 1:20 togive values, which are linear with respect to a standard curve of mouseIgE. Results are expressed in μg/ml. Afu-specific IgGl is measured byELISA using 96-well plates coated with Afu allergen extract. Antibody isdetected with HRP-labeled anti-mouse IgGl. Results are expressed asrelative absorbance units (OD450).

[0454] Endogenous Mouse SP-D and SF-A in the Lung

[0455] Immediately after humane sacrifice by CO₂ asphyxiation,broochoalveolar lavage is performed with 3×1 ml PBS, which are pooledand the volumes adjusted by addition of PBS to 4 ml for all samples andcentrifuged to remove cells. SP-D and SP-A are measured by ELISA usingpolyclonal antibodies raised against recombinant mouse SP-D and SP-A(kindly provided by Dr. P. Lawson). These antibodies are shown not tocross-react with human SP-D or SP-A and are specific for mouse SP-D orSP-A, respectively. Results are expressed as μg/ml of BAL.

[0456] Intracellular Cytokine Staining.

[0457] After treatment, mice are humanely sacrificed by CO₂ asphyxiationand their spleens removed and homogenized in PBS. The homogenate isfiltered and red blood cells lysed with ammonium chloride lysing reagent(BD Pharmingen) and fixed with 4% (v/v) paraformaldehyde for 20 mins.The cells are washed with PBS supplemented with 3% (v/v) heatinactivated fetal calf serum with 0.1% (w/v) sodium azide (FSB),re-suspended in 10% DMSO (v/v) in FSB and stored at −80° C. Cells arepermeabilized with Cytoperm wash buffer (CPB, BD Biosciences, Cowley,UK) for 15 mins at 4° C. and aliquots of 10⁶ cells are blocked byincubation for 30 mins at 4° C. with CPB supplemented with 50 μg/ml ratIgG. Intracellular cytokines are stained with 1 μg PE-conjugatedanti-mouse cytokine monoclonal antibody (BD Biosciences) incubated for60 mins at 4° C. The cells are washed with CPB followed by FSB andre-suspended in 500 1 FSB. Flow cytometry is performed with a FACScanflow cytometer (Beckton Dickinson, Mountain View, Calif.) usingCellQuest software. Data are collected for 20000 cells. The average FSCof spleen cells is 100 in all cases. Stained cells (FSC>100, FL2>100)are gated and the proportion of these cells staining intensely for PE(PE>1000) is calculated. Results are expressed as the % intenselystained cells after subtraction of background fluorescence for unstainedcells incubated with rat IgG (% PE>1000).

[0458] Lung Histology

[0459] Immediately after treatment, the lungs of 2-4 mice from eachtreatment group are fixed in 10% (v/v) neutral buffered formalin andsent for independent analysis. Lungs are embedded in paraffin, sectionedand stained with hematoxylin and eosin. The slides are evaluated forperibronchial inflammation and scores are assigned on a scale of 0-4,corresponding to a score of normal to severe, respectively [Sur S, WildJ S, Choudhury B K et al. Long term prevention of allergic lunginflammation in a mouse model of asthma by CpG oligodeoxynucleotides. JImmunol 1999; 162:6284-93.].

[0460] Whole Body Plethysmography.

[0461] In this study, airway hyperresponsiveness is measured usingunrestrained whole body plethysmography [Hamelmann E, Schwarze J, TakedaK et al. Noninvasive measurement of airway responsiveness in allergicmice using barometric plethysmography. Am J Respir Crit Care Med 1997;156:766-75] with a four-chamber system (Buxco, Sharon, Conn.). Mice arefirst challenged with intranasal antigen and allowed to recover for 2hours before being placed into the chambers and their breathingmonitored for 10 mins. When acclimatized, their baseline response ismeasured for 5 mins. The mice are then subjected to 1 min of aerosolisedPBS, followed by progressively increasing doses of methacholine (5, 10,20, 30, 40 mg/ml PBS). Responses are recorded for 5 mins in every casewith a short interval between to allow return to baseline Penh.

[0462] Each group contained 4-8 mice. Results are presented as theaverage % elevation in Penh over baseline after a challenge ofmethacholine.

[0463] Statistics

[0464] Results are the average for 4-8mice/group and error bars are SEM.Significance is determined by Student's 2-tailed t-test. Significance isaccepted for P<0.05.

[0465] Example 2 Results

[0466] Results on treatment of sensitised mice with recombinant SP-D areshown in FIGS. 2, 3, 4 and 5.

[0467] Serum IgE, Afu-Specfic IgGl and Peripheral Blood Eosinophilia areReduced by rSPD(n/CRD) in a Different Genetic Background

[0468] To determine if treatment with rSPD(n/CRD) is effective in adifferent genetic background an ABPA model is established in C57BL/6mice and to determine if rSPD(n/CRD) could modulate allergichypersensitivity during allergen challenge the sensitised mice are firstchallenged with 10 μg Afu lwcf and treated 1 hour later. Serum IgEmeasured 3 days after treatment with 5 daily doses of 10 μg rSPD(n/CRD),given intranassaly to allergen challenged mice is significantly reduced(P<0.001) and this reduction is maintained after re-challenge with 3daily doses of 10 μg Afu lwcf given the following week (FIG. 2A). Asimilar significant reduction is also measured in Afu-specific IgGl(FIG. 2B) and peripheral blood eosinophilia measured alter treatment ofallergen challenged mice and re-challenge with allergen alone thefollowing week (FIG. 3).

[0469] Treatment with rSPD(n/CRD) Results in Elevation in IL-12 andIFN-γ and a Reduction in IL-4

[0470] The reduction in IgE and peripheral blood eosinophilia suggests asystemic modulation at the cytokine level and these cytokines aremeasured by intracellular staining. IL-12 (FIG. 4A) measured in thespleen, 1 day alter treatment for 2 days with 10 μg rSPD(n/CRD), givenintranasally to allergen challenged mice is significantly reduced(P<0.05) as is IFN-γ (FIG. 4B). Measurement of IL-4 showed a decrease tothe level measured in non-sensitised mice (FIG. 4C). The same treatmentwith rhSP-A (recombinant human surfactant protein A, SP-A) did notproduce these effects.

[0471] Treatment with rSPD(n/CRD) Results in Reduced AirwayHyperresponsiveness.

[0472] Mice treated with 4 daily doses of 10 μg rSPD(n/CRD) given 1-2hours after allergen challenge showed a significant reduction (P<0.05)in airway hyperresponsiveness on re-challenge with allergen 3 days aftercompletion of treatment (day7) in all methacholine doses tested (FIG.5A). Mice treated with 10 μg rhSP-A (recombinant human surfactantprotein A) in the same way did not show a significant reduction in AHR(FIG. 5B).

[0473] Treatment with rSPD(n/CRD) Results in Reduced Lung Inflammation

[0474] Of the sensitised mice in this study 4 of the 6 PBS treated micehad a score of 2+ and of the 5 in the rSPD(n/CRD) treatment group 4 hada score of 1. The score for non-sensitised mice is 0. Lung sectionsillustrate the reduction in cellular infiltration into the lungs (FIG.6).

[0475] Endogenous Mouse SP-D and SP-A.

[0476] Endogenous levels of SP-D measured in the BAL of allergic miceare elevated 6 fold from a level of 0.25±0.015 μg/ml in BAL from normalmice to 1.4±0.151 μg/ml, while no difference is found for the level ofendogenous SP-A which is measured at 1.3±0.1 μg/ml in normal, sensitisedand treated mice BAL. Treatment with 5 daily doses of 10 μg rSPD(n/CRD)did not produce any change in the level of endogenous SP-D or SP-Alevels.

Example 3 Modulation of Allergic Hypersentivity in a Murine Model ofAllergic Hypersensitivity to House Dust Mite (Dermatophagoidespteronyssinus)

[0477] Derp/IL-2/Lung

[0478]FIG. 7A shows that intranasal treatment with rSP-D (N/CRD) resultsin an enhancement in the IL-12 response to allergen challenge in thelungs of Der p allergic mice

[0479] An analysis of IL-12 in the lung homogenates of Der p sensitisedmice after treatment measured by intracellular cytokine stainingfollowed by FACS analysis for the percentage of highly stained cells(PE>1000) positive for IL-12. PBS=non-sensitised mice treated with PBS.HP=sensitised mice treated with PBS. HR=sensitised mice treated with 10μg rSP-D (N/CRD). Sensitised mice were first challenged with 50AU of Derp allergen extract given intranasally. Treatment was by intranasalinstillation of 10 μg rSP-D (N/CRD) given shortly after challenge.Challenge and treatment were repeated on 4 consecutive days. Mice werere-challenged with Der p alone 4 days after treatment and sacrificed thefollowing day.

[0480] Derp/IL-2/Spleen

[0481]FIG. 7B shows evidence that intranasal treatment with rSP-D(N/CRD) results in an enhancement in the IL-12 response to allergenchallenge in the spleen of Der p allergic mice.

[0482] An analysis of IL-12 in the spleen homogenates of Der psensitised mice after treatment measured by intracellular cytokinestaining followed by FACS analysis for the percentage of highly stainedcells (PE>1000) positive for IL-12. PBS=non-sensitised mice treated withPBS. HP=sensitised mice treated with PBS. HR=sensitised mice treatedwith 10 μg rSP-D (N/CRD). HB=sensitised mice treated with 10 μg BSA.Sensitised mice were first challenged with 50AU of Der p allergenextract given intranasally. Treatment was by intranasal instillation of10 μg rSP-D (N/CRD) given shortly after challenge. Challenge andtreatment were repeated on 4 consecutive days. Mice were re-challengedwith Der p alone 4 days after treatment and sacrificed the followingday. (n=4-8/group. 1.7× elevation.).

[0483] Derp/INF-α/Spleen

[0484]FIG. 7C shows evidence that intranasal treatment with rSP-D(N/CRD) results in an enhancement in the IFN-γ response to allergenchallenge in the spleens of Der p allergic mice.

[0485] An analysis of IFN-y in the spleen homogenates of Derp sensitisedmice after treatment measured by intracellular cytokine stainingfollowed by FACS analysis for the percentage of highly stained cells(PE>1000) positive for IFN-γ. PBS non-sensitised mice treated with PBS.HP=sensitised mice treated with PBS. HR=sensitised mice treated with 10μg rSP-D (N/CRD). Sensitised mice were first challenged with 50AU of Derp allergen extract given intranasally. Treatment was by intranasalinstillation of 10 μg rSP-D (N/CRD) given shortly after challenge.Challenge and treatment were repeated on 4 consecutive days. Mice werere-challenged with Der p alone 4 days after treatment and sacrificed thefollowing day. (n=4-8/group. P<0.05. 2.7×elevation).

[0486] Derp/TNF-α/Spleen

[0487]FIG. 7D shows evidence that intranasal treatment with rSP-D(N/CRD) results in an enhancement in the TNF-α response to allergenchallenge in the spleens of Der p allergic mice.

[0488] An analysis of TNF-α in the spleen homogenates of Der psensitised mice alter treatment measured by intracellular cytokinestaining followed by FACS analysis for the percentage of highly stainedcells (PE>1000) positive for TNF-α. PBS non-sensitised mice treated withPBS. HP=sensitised mice treated with PBS. HR=sensitised mice treatedwith 10 μg rSP-D (N/CRD). Sensitised mice were first challenged with50AU of Der p allergen extract given intranasally. Treatment was byintranasal instillation of 10 μg rSP-D (N/CRD) given shortly afterchallenge. Challenge and treatment were repeated on 4 consecutive days.Mice were re-challenged with Der p alone 4 days after treatment andsacrificed the following day. (n=4-8/group. 1.5× elevation).

Example 4 Effect of Treatment with rSPD(n/CRD) on AirwayHyperresonsiveness in a Murine Model of Allergic Hypersensitivity toHouse Dust Mite (Dermatophagoides pteronyssinus)

[0489] Allergic asthma is characterised by periods of airwayhyperresonsiveness (AHR). AHR is known to alter breathing patternsincluding an extension of the expiration time, This change can bequantified by the measurement of enhanced expiratory pause (Penh).

[0490] In this study, airway hyperresponsiveness is measured usingunrestrained whole body plethysmography with a 4 chamber system(Buxco,Sharon,Conn.,USA). A constant bias flow of air is drawn throughthe system in order to prevent accumulation of CO₂. As the mice breathein the mouse chamber, pressure fluctuations are measured by transducersand compared to a reference chamber. These fluctuations are produced bychanges in thoracic volume during the breathing cycle.Bronchoconstriction produces differences in the shape of the pressureexcursions and is most pronounced during expiration. These shape changescan be quantified by the algorithm for enhanced pause (Penth), whichrepresents a difficulty in expiration and is a characteristic ofbreathing in asthmatics.${Penh} = {\frac{{Te} - {Tr}}{Tr} \times \frac{PEP}{PIP}}$

[0491] Te is the expiratory time, Tr is the relaxaion time, PEP is thepeak expiratiory pressure and PIP is the peak inspiratory pressure.

[0492] AHR is provoked by exposing the mice to nebulised methacholine,in situ. Methacholine (Acetyl-β-methylcholine chloride) is ametabolically stable analogue of the neurotransmitter acetylcholine andcauses contraction of smooth muscle resulting in provokedbronchoconstriction. The provoked AER of asthmatic mice is measured byan increase in Penh relative to normal mice.

[0493] Methods

[0494] Sensitisation. Female C57BL/6 mice are sensitised by 4 weeklyi.p. injections of standerdised Der p extract (Greer Labs., USA) withalum.

[0495] Induction of airway inflammation and treatment. Sensitised miceare given 50 allergy units of Der p extract in PBS, intra nasally (i.n),preceded by i.n. administration of PBS or 10 μg rSPD(n/CRD), referred toas sequential co-administration, on 4 successive days. Animals areallowed to recover for at least 30 mins before the second i.n.administration.

[0496] Whole body plethysmography. Mice re placed into the four chambersand their breathing monitored for 10 mins. When acclimatised, theirbaseline response is measured for 5 mins. Next, they are subjected to 1mm of aerosolised PBS, followed by progressively increasing doses ofmethacholine. Responses are recorded for 5 mins in every case with ashort interval between to allow return to baseline.

[0497] Statistics. Each group contains 4-8 mice. Penh is expressed asthe average % increase over the baseline value for the mice in eachgroup. Error bars are ±SEM. Significance is determiner by pairedt-tests.

[0498] Example 4 Results

[0499] Treatment with rSPD(n/CRD) produces a significant decrease(p<0.001) in AHR as measured by the Penh response in whole bodyplethysmography, at the end of the treatment period (FIG. 8A; 20 mg/mlmethacholine). This significant decrease was maintained when mice arere-challenged with allergen 4 days after treatment (p<0.002) as shown inFIG. 8B (methacholine dose response with re-challenge).

[0500] This study on a mice model of house dust mite allergy shows forthe first time that intra-nasal administration of rSPD(n/CRD)significantly reduces airway hyperresponsiveness, which is a majorfeature of asthma. It provides evidence for the usefulness ofrSPD(n/CRD) in treating the symptoms of repiratory allergichypersensitivity reactions including allergic asthma.

[0501] It is of significance that rSPD(n/CRD) is able to reduce AHR evenin the presence of sustained allergen exposure as demonstrated by itsefficacy during the sequential co-administration with Der p allergen.This mirrors the situation encountered by seasonal allergic subjects orthose constantly exposed to aeroallergens, which is a major problem inhouse dust mite allergy.

[0502] The most remarkable finding is that treatment with rSPD(n/CRD)has a long term effect on the underlying mechanisms as shown by thesignificantly reduced AHR on rechallenge with allergen alone. Thustreatment with rSPD(n/CRD) provides an effective desensitisationstrategy for persons suffering from allergy and allergic asthma.

Example 5 Effect of Recombinant SP-D on Knock-Out Mice

[0503] The following three Examples describes experiments in which SP-Dfunction is replaced in SP-D knock-out mice using recombinant humanSP-D.

[0504] SP-D knock-out mice which express no SP-D, have an increasednumber of macrophages, many of which are abnormal in appearance. Thereis an excess of surfactant phospholipid in the alveolar space,indicating a chronic low grade inflammatory process in the absence ofSP-D. These mice subsequently develop lung injury and fibrosis andemphysema and provide a model for the contribution made to lung injuryand inflammation of SP-D deficiency (as seen in the premature infant andin smokers, patient groups with congenital and acquired SP-D deficiency,respectively). The effect on this inflammatory process of treatment withrecombinantly expressed truncated fragment, human rSP-D (N/CRD), isassessed in SP-D knock-out mice.

[0505] SP-D knockout mice are produced as described in Botas, C., etal., Altered surfactant homeostasis and alveolar type II cell morphologyin mice lacking surfactant protein D. Proc Natl Acad Sci USA, 1998.95(20): p. 11869-74. Recombinant human SP-D is produced as describedabove.

[0506] Recombinant SP-D or BSA control is administered intranasally tomice from the age of 6 weeks. A total of 7 doses of 30 micrograms isgiven over 6 weeks, until the animals are 12 weeks old. Four mice resacrificed after 3 weeks of treatment and underwent bronchoalveolarlavage for cell counts of alveolar macrophage number and phospholipidestimation. The experiments as described are repeated three times.

[0507] These Examples demonstrate that rSPD(n/CRD) may be used as aneffective treatment for inflammatory lung disease.

Example 6 Effect of Recombinant SP-D on Knock-Out Mice as Assayed byAlveolar Macrophage Numbers

[0508] The number of macrophages stained with Malachite green or crystalviolet is counted in a haemocytometer and the morphology of themacrophages is assessed after cytospin.

[0509]FIG. 9 shows the number of alveolar macrophages counted inuntreated mice and demonstrates that the number of cells isapproximately three times those counted in wild type mice at 6 weeks andby 9 weeks and 12 weeks is almost four times wild-type. The number ofmacrophages is maintained in untreated knock-out mice, so that there arecomparable numbers of cells harvested from the lavage at 16 weeks.However, alter treatment with rSP-D (N/CRD), the number of alveolarmacrophages in SP-D knock-out BAL is reduced by approximately 50%, tolevels roughly twice those seen in wild-type mice of comparable age.FIG. 9 also shows that alveolar macrophage numbers are increased in SP-Ddeficient compared to wild-type mice at 15 weeks.

[0510] The effect of treatment with rSP-D on the number of alveolarmacrophages isolated from BAL fluid is shown. In contrast with treatmentwith rhSP-A, BSA or PBS, the number of macrophages is significantlydecreased in rSP-D treated SP-D deficient mice at 9, 12, and 15 weeks.Macrophage numbers in rSP-D treated animals remain higher than in wildtype mice at all ages.

[0511]FIG. 10A shows a cytospin of cells in broncho-alveolar lavage.Cells are stained with Malachite green (upper panels) or crystal violet(lower panels). It is clear that there are fewer alveolar macrophages inrSP-D (N/CRD) treated mice and they have a more normal appearance withvery few enlarged foamy macrophages visible per high power field,compared to untreated or BSA treated mice.

[0512]FIG. 10B shows a cytospin of cells in broncho-alveolar lavage.Cells from SP-D deficient mice are large, foamy and oftenmultinucleated. In contrast from BSA or SP-A treated mice, alveolarmacrophages from rSP-D treated mice are more frequently normal inappearance, with fewer enlarged and foamy cells. This effect isquantified by forward and side scatter flow cytometry.

Example 7 Effect of Recombinant SP-D on Knock-Out Mice as Assayed bySurfactant Phospholipid Analysis

[0513] Phospholipid extraction is carried out from BAL and thephospholipid quantified by Bartlett phosphorus assay, as described inBartlett, G. R., Phosphorus assay in column chromatography. J. Biol.Chem., 1959. 234: p. 466-468.

[0514] Results are shown in FIG. 11. There is no detectable differencein phospholipids assayed after 3 weeks of treatment in mice which hadreached 9 weeks of age, whereas the amount of phospholipid recoverablein the lavage after 6 weeks of treatment is much reduced.

[0515] Further results relating to total phospholipid and proteinmeasurements in cell free BAL are shown in FIG. 11B. There is a markedincrease in BAL phospholipid levels in SP-D deficient mice by 6 weeks ofage. There is no difference in phospholipid levels in mice receiving twoweeks of treatment with rSP-D from age four weeks compared to untreatedsix-week old mice

[0516] The same mice as used in the alveolar macrophage count experiment(FIG. 9) which are treated from age six weeks for three or six weeks arealso assayed for cell free BAL phospholipid. There is no difference inBAL phospholipids after three weeks of treatment (sacrifice age nineweeks) compared to untreated nine-week old controls. However micetreated for six weeks (sacrificed for assay age 12 weeks) show a 50%reduction in excess phospholipid compared to untreated twelve week oldcontrols (P<0.05). There is no significant effect of treatment on lavagetotal protein levels at any age.

[0517] Plasma cholesterol levels are determined in SP-D deficient mice,as shown in FIG. 17. This figure compares plasma cholesterol in mMbetween wild type mice and SP-D deficient mice. A paired test wasconducted, with a hypothesised difference of 0. The mean difference is0.988, the DF is 8, the T-value is −2.885 and the P-value is 0.0204.

Example 8 Effect of Recombinant SP-D on Knock-Out Mice as Assayed byChemokine Levels

[0518] To evaluate possible mechanisms to explain the findings ofreduced alveolar macrophage number, an apparently rSP-D specific effect,total RNA is isolated from rSP-D (N/CRD) treated knock-out mouse lungsand untreated controls and the levels of mRNA for a number ofchemotactic factors assayed by ribonuclease protection assay using themCK-5 multi probe template set from Pharminogen in accordance with themanufacturer's instructions. Ribonuclease protection assays areconducted on total lung RNA.

[0519] The results are normalised across samples to check for equalloading of total lung RNA using the mRNA signal for L32 and GAPDH. Eachsample is loaded in duplicate in 2 concentrations, using 3 mice for eachgroup.

[0520] The results are shown in FIG. 12. It can be seen that there aresignificant differences in baseline mRNA levels between SP-D knock-outmice and wild-type mice. Specifically, levels of Eotaxin, the specificeosinophil chemotactic factor are increased by 100% in knock-out mice ascompared to wild type. Furthermore, this is decreased by treatment withrSP-D (N/CRD). These observations are statistically significant at the5% level using t-test for unpaired data. MCP-1 mRNA levels are increased6 fold in knock-out mice as compared to wild type, and this is alsodecreased by treatment with rSP-D (N/CRD). A similar effect is observedon MIP-1 alpha levels and on MIP 2 except that there is no significanteffect of rSP-D (N/CRD) treatment on MIP-2 mRNA levels. A finalobservation is that TCA-3 levels are increased 4 fold in knock-outversus wildtype mice, but treatment with rSP-D (N/CRD) has no effect onthe increased expression of TCA-3.

Example 9 rSP-D (N/CRD) Promotes Clearance of Apoptotic AlveolarMacrophages

[0521] The process of regulated cell death by apoptosis avoids cellnecrosis and consequent release of pro-inflammatory intracellularcontents. Given the excess of alveolar macrophage numbers in SP-Dknock-out mice, the number of cells undergoing apoptosis in knock-outmice is measured and compared to wild type.

[0522] We hypothesised that lung injury mediated by the presence oflarge numbers of alveolar macrophages is due in part to impairedclearance of apoptotic alveolar macrophages with consequent triggeringof an inflammatory response due to increased alveolar macrophagenecrosis.

[0523] Flow Cytometry and Detection of Apoptotic and Necrotic Cells

[0524] Alveolar macrophages are isolated from mice by bronchoalveolarlavage as described above.

[0525]FIG. 13A shows a representative result of forward scatter (cellsize) and side scatter (granularity) flow cytometry in SP-D deficientmice compared to wild-type mice. Ten thousand cells are counted in eachmouse from each group (n=6 per group). The histograms show a populationof larger and more granular cells in SP-D deficient mice consistent withthe cytospin appearances of enlarged foamy macrophages in SP-D deficientmice compared to those isolated from wild-type mice.

[0526] The effect of rSP-D treatment on the forward and side scatter ofthe cell population is shown using representative histogram overlays,and indicates a cell population with fewer abnormally large and granularalveolar macrophages, consistent with the cytospin appearances.Treatment with rhSP-A, PBS or BSA had no effect on the shape of forwardand side scatter histograms.

[0527] Apoptosis and Necrosis in Alveolar Ma Crop Hages

[0528] Alveolar macrophages are isolated from mice by bronchoalveolarlavage as described above and assayed for apoptosis and necrosis bymeasuring binding of FITC labelled-Annexin V (which binds to surfacephosphatidyl serine exposed on apoptotic cells) and Propidium Iodide(which binds necrotic cells) by FACS analysis.

[0529] As shown in FIGS. 13B, 13C and 13D, there is a large differencein the number of apoptotic and necrotic macrophages in lavage from SP-Dknock-out mice compared to wild-type.

[0530] Annexin V and PI Staining of Freshly Isolated AlveolarMacrophages

[0531]FIG. 13B shows the typical patterns of annexin V and PI stainingof macrophages from SP-D deficient mice compared to wild-type mice.Consistently, the number of macrophages, staining with annexin V, issignificantly higher in SP-D deficient mice compared to wild type, andthere is a three to four fold increase in the number of cells thatstained with both annexin V and PI in SP-D deficient mice. The arbitrarycut off shown in the Figure, dividing the cell population intoquadrants, allows calculation of the percentages of cells staining withPI or annexin V alone in each case.

[0532] Co-Labelling of Annexin V and/or PI Positive Cells withFITC-Labelled rSP-D.

[0533]FIG. 13C shows the extent of co-labelling of FITC-labelled rSP-Dwith annexin V and/or PI positive cells. Results are representative ofthree experiments. The dot plots show preferential binding of FITC-rSP-Dto early apoptotic (annexin V positive) and necrotic (PI positive)cells. Overall, a higher proportion of cells isolated from knock-outmice are bound by FITC-rSP-D, compared to wild-type (45+/−6%versus15+/−1.8%). Of the small number of annexin V positive cells inwild-type mice, 80+/−6% are bound by FITC-rSP-D, compared with only1+/−0.5% of annexin V negative cells. In knock-out mice, 55+/−4% ofannexin V positive AM bound FITC-rSP-D, compared with only 3.5+/−1.7% ofannexin V-negative cells. All cells which stained with PI from wild-typeor knock-out mice are bound by FITC-rSP-D. Thus, there is a low level ofbinding of FITC-rSP-D to healthy cells (PI negative, annexin V negative)from wild-type mice and knockout mice compared to the binding to annexinV or PI positive cells. Co-incubation of freshly isolated cells withFITC-annexin V and unlabelled rSP-D did not significantly affect annexinV binding (not shown), indicating that there is no direct interactionbetween annexin V and rSP-D, nor any competition by rSP-D of annexin Vbinding to its phosphatidylserine ligand.

[0534] Analysis and Treatment with rSP-D

[0535]FIG. 13D shows that SP-D deficient mice have a ten fold increasein the proportion of apoptotic alveolar macrophages in bronchoalveolarlavage compared to wild-type mice (knock-out 74%+/−8% vs 6%+/−2% inwildtype) and a six fold increase in necrotic alveolar macrophages(25%+/−8% in knock-out versus 4+/−2% in wildtype). Intranasalreplacement therapy for 3-6 weeks with r SP-D (N/CRD) increasesclearance of apoptotic and necrotic alveolar macrophages from thealveolar space in SP-D deficient animals, whereas similar treatment withbovine serum albumin or surfactant protein A (SP-A) has no effect.

[0536] The effect of treatment with rSP-D on the percentages of cellsstaining with either PI or annexin V is also shown in the Figure.

[0537] The percentage of apoptotic macrophages are 25%+/−4% in rSP-D(N/CRD) treated mice versus 76%+/−10% in controls. Treatment with rSP-D(N/CRD) reduces the percentage of necrotic macrophages in lavage of SP-Dknockout mice (around 25%) to levels comparable with wild type (5%versus 4%). The overall effect of treatment with rSP-D (N/CRD) is todecrease the absolute number of alveolar macrophages in the lung, alongwith levels of pro-inflammatory cytokines and excess surfactantphosholipids by 50%.

[0538] There is no significant effect of treatment with rhSP-A, BSA, orPBS on the percentage of cells staining with annexin V and/or PI. Bycontrast, rSP-D treatment resulted in a significant reduction in thenumber of annexin V and/or PI positive cells in the BAL of SP-Ddeficient mice, though the number of annexin V and/or PI positive cellsis still significantly higher than wild-type.

[0539] Modulation of GM-CSF Levels in Bronchoalveolar Lavage

[0540] Modulation of GM-CSF levels in bronchoalveolar lavage are shownin FIG. 14. As can be seen, the concentration of GM-CSF inbronchoalveolar lavage of wild-type mice is about 50 g/ml, compared toabout 185 pg/ml in SP-D knock-out mice. Treatment of such knock-out micewith rSP-D (N/CRD) reduces the levels of GM-CSF to about 120 g/ml.

[0541] Results from flow cytometry and biochemical analyses areconsistent with lung histology and cytospin examination of alveolarmacrophages harvested from bronchoalveolar lavage.

[0542] Confocal Microscopy

[0543]FIG. 15 shows confocal microscopy of macrophages from wild-type(panels A and B) and knock-out mice (panels C and D), stained withFITC-dUTP (green). Cells in advanced apoptosis stain green and cells inearly apoptotis are identified by characteristic punctate staining ofend labelled DNA fragments.

[0544] Detection of Phagocytosis of Orange Stained Necrotic andApoptotic Macrophages by Green Stained Freshly Isolated Macrophages

[0545] Cells are isolated from knock-out mice as described above andincubated with Orange cell tracker dye overnight at room temperature.Colabelling with Annexin V-FITC confirms that 80-90% of these cells aredouble labelled with orange dye and are Annexin V positive. Freshlyisolated cells are incubated with Cell tracker Green in accordance withthe manufacturer' instructions. Many cells fail to stain with Celltracker green which depends on live cells (FIG. 16, lower left quadrant,panel A), and a proportion of cells were successfully labelled (FIG. 16,lower right quadrant, panel A)

[0546] Panel A shows the result of immediate mixing of orange and greencell populations. Using the gate indicated to exclude unstained cellsand background double fluorescence (indicated on panel B), panels C andD show double labelling after coincubation of cells for 15 minutes at 37degrees without SP-D (panel C) and with SP-D (panel D) at 4 mcg/ml. Thenumber of double labelled cells is enhanced in the presence of SP-D by80%.

[0547] These results in SP-D knock-out mice reveal a critical role forSP-D in regulating clearance of apoptotic and necrotic macrophages andindicate that rSP-D (N/CRD) based therapy provides a novel approach in aspectrum of lung disease involving alveolar macrophage mediatedinflammatory processes and other diseases.

[0548] However, the shown data relating to involvement of SP-D in theregulation and clearance of apoptotic cells also have implications forabnormalities of development (e.g., abnormal post natal lung developmentin neonatal lung disease following premature birth), remodelling ofinflamed tissues, cellular proliferation and cancer.

Example 10

[0549] Binding of SP-D and rSPD(n/CRD) (referred to as recombinantfragment SP-D, rSPD(n/CRD)) to DNA is tested in a variety of ways. In afirst assay electron microspcopy is used to demonstrate complexesbetween DNA and SP-D, and DNA and rf-SP-D. See FIG. 18. The imagesobtained by electron microscopy demonstrate that SP-D and rSPD(n/CRD)bind DNA effectively.

[0550] In a second assay, shown in FIG. 19, binding between rSPD(n/CRD)and the carbohydrate mannan is competed by deoxyribonucleotides,suggesting that SP-D binds to DNA via its carbohydrate binding capacity.The deoxyribonucleotides effectively compete for binding to SP-D withimmobilised mannan, showing that the nucleiotides are bound in solution.

[0551] In a third assay, shown in FIG. 20, alveolar macrophages isolatedfrom the SP-D knock out mice (mainly apoptotic), were incubated withindicated concentrations of rSPD(n/CRD) followed by propidium iodide,and the red colour propidium iodide that was bound to the cell wasdetermined by FACS analysis. In the presence of 4 and 20 μg/mLrSPD(nJCRD), 11.4 and 2.1% of the cells contained propidium iodide,respectively, compared with 16.8% in the absence of the protein. Theseresults show that rSPD(n/CRD) binds DNA on the apoptotic cells.

[0552] To obtain evidence of the defect in the clearance of DNA in vivo,we isolated alveolar macrophages from SP-D knockout mice (SP-D^((−/−)))and compared their ability to take up DNA with that of wild type andSP-A knockout mice (SP-A^((−/−))). The data (FIG. 21) show that alveolarmacrophages isolated from SP-D deficient mice were poor (4.0%) inclearing DNA compared with that of wild type (10.5%) or SP-A deficientmice (13.2). These results suggest that SP-D plays an important role inDNA clearing pathway in the lung.

[0553] References

[0554] Platts-Mills, T. A., and M. D. Chapman. 1987. Dust mites:immunology, allergic disease, and environmental control. J Allergy ClinImmunol. 80:755-75.

[0555] Platts-Mills, T. A., B. B. Mitchell, M. D. Chapman, and P. W.Heymann. 1987. Dust mite allergy: its clinical significance. Hosp Pract(Off Ed). 22:91-3, 97-100.

[0556] Pollart, S. M., M. D. Chapman, and T. A. Platts-Mills. 1987.House dust sensitivity and environmental control. Prim Care. 14:591-603.

[0557] Arruda, L. K., B. J. Mann, and M. D. Chapman. 1992. Selectiveexpression of a major allergen and cytotoxin, Asp f I, in Aspergillusfumigatus. Implications for the immunopathogenesis ofAspergillus-related diseases. J Immunol. 149:3354-9.

[0558] Cai, G. Z., G. L. Griffin, R. M. Senior, W. J. Longmore, and M.A. Moxley. 1999. Recombinant SP-D carbohydrate recognition domain is achemoattractant for human neutrophils. Am J Physiol. 276:L131-6.

[0559] Hickling, T. P., H. Bright K. Wing, D. Gower, S. L. Martin, R B.Sim, and R Maihotra. 1999. A recombinant trimeric surfactant protein Dcarbohydrate recognition domain inhibits respiratory syncytial virusinfection in vitro and in vivo. Eur J Immunol. 29:3478-84.

[0560] Kishore, U., L. E. A. Leigh, P. Eggleton, P. Strong, M. V.Perdikoulis, A. C. Wiffis, and K. B. Reid. 1998. Functionalcharacterization of a recombinant form of the C-terminal, globular headregion of the B-chain of human serum complement protein, C1q. Biochem J.333:27-32.

[0561] Madan, T., U. Kishore, A. Shah, P. Eggleton, P. Strong, 3. Y.Wang, S. S. Aggrawal, P. U. Sarma, and K. B. Reid. 1997. Lung surfactantproteins A and D can inhibit specific IgE binding to the allergens ofAspergillus fumigatus and block allergen-induced histamine release fromhuman basophils. Clin Exp Immunol. 110:241-9.

[0562] Madan, T., U. Kishore, M. Singh, P. Strong, H. Clark, B. M.Hussain, K. B. Reid, and P. U. Sarma. 2001. Surfactant proteins A and Dprotect mice against pulmonary hypersensitivity induced by Aspergillusfumigatus antigens and allergens. J Clin Invest. 107:467-75.

[0563] Madan, T., U. Kishore, M. Singh, P. Strong, E. M. Hussain, K. B.Reid, and P. U. Sarma. 2001. Protective role of lung surfactant proteind in a murine model of invasive pulmonary aspergillosis. Infect Immun.69:2728-3 1.

[0564] Awasthi S, Coalson J J, Yoder B A, Crouch E, King R I.Deficiencies in lung surfactant proteins A and D are associated withlung infection in very premature neonatal baboons. Am J Respir Crit CareMed. 2001 February;163(2):389-97 .

[0565] Wert S E, Yoshida M, LeVine A M, Ikegami M, Jones T, Fisher J H,Korfhagen T R, Whitsett I A. Increased metalloproteinase activity,oxidant production, and emphysema in surfactant protein Dgene-inactivated mice. Proc Natl Acad Sci USA. 2000 May23;97(11):5972-7.

[0566] Madan, T., et al., Surfactant proteins A and D protect miceagainst pulmonary hypersensitivity induced by Aspergillus fumigatusantigens and allergens. J Clin Invest, 2001. 107(4): p.467-75.

[0567] The following numbered citations are referred to in the sectionon “Surfactant Replacement Therapy”:

[0568] 1 .Jobe A. Surfactant treatment for respiratory distresssyndrome. Respir Care 1986;31 (6):467-476.

[0569] 2.Berry D. Neonatology in the 1990's: surfactant replacementtherapy becomes a reality. Clin Pediatr 1991;30(3):167-170.

[0570] 3.Avery M E, Mead I. Surface properties in relation toatelectasis and hyaline membrane disease. Am J Dis Child1959;97:517-523.

[0571] 4.von Neergard K. Neue Auffassungen uber einen Grundbegriff derAtemmechanik: die Retraktionskraft der Lunge, abhangig von DrOberflächenspannung in den Alveolen. Z Ges Exp Med 1929;66:373.

[0572] 5.Hallman M, Teramo K, Ylikorkala 0, Merritt T A. Naturalsurfactant substitution in respiratory distress syndrome. J Perinat Med1987;15:463-468.

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[0574] 7.Horbar J D, Wright B C, Onstad L. Decreasing mortalityassociated with the introduction of surfactant therapy: an observationalstudy of neonates weighing 601 to 1300 grams at birth. Pediatrics1993;92(2):191-196.

[0575] 8.Lang M I, Hall R T, Reddy N S, Kurth C G, T A Merritt. Acontrolled trial of human surfactant replacement therapy for severerespiratory distress syndrome in very low birth weight infants. JPediatr 1990;116(2):295-300.

[0576] 9.Hoekstra R E, Jackson J C, Myers T F, Frantz ID 111, Stern M E,Powers W F, et al. Improved neonatal survival following multiple dosesof bovine surfactant in very premature neonates at risk for respiratorydistress syndrome. Pediatrics 1991;88(1):10-18.

[0577] 10.Kattwinkel I, Bloom B T, Delmore P, Davis C L, Farrell E,Friss H, et al. Prophylactic administration of calf lung surfactantextract is more effective than early treatment of respiratory distresssyndrome in neonates of 29 through 32 weeks' gestation. Pediatrics1993;92(1):90-98.

[0578] 11.Merritt T A, Hallman M, Berry C, Pohjavuori M, Edwards D K,Jaaskelainen J, et al. Randomized, placebo-controlled trial of humansurfactant given at birth versus rescue administration in very low birthweight infants with lung immaturity. J Pediatr 1991;118(4):581-594.

[0579] 12.Dunn M S, Shennan A T, Zayack D, Possmayer F. Bovinesurfactant replacement therapy in neonates of less than 30 weeks'gestation: a randomized controlled trial of prophylaxis versustreatment. Pediatrics 1991;87(3):377-386.

[0580] 13.Long W, Thompson T, Sundell H, Schumacher R, Volberg F,Guthrie R, et al. Effects of two rescue doses of a synthetic surfactanton mortality rate and survival without bronchopulmonary dysplasia in700- to 1350-gram infants with respiratory distress syndrome. I Pediatr1991;118(4):595-605.

[0581] 14.Liechty E A, Donovan B, Purobit D, Gilhooly I, Feldman B,Noguchi A, et al. Reduction of neonatal mortality after multiple dosesof bovine surfactant in low birth weight neonates with respiratorydistress syndrome. Pediatrics 1991;88(1):19-28.

[0582] 15.Long W, Corbet A, Cotton R, Courtney S, Mc Guiness 0, WalterD, etal. A controlled trial of synthetic surfactant in infants weighing1250 g or more with respiratory distress syndrome. N Engl J Med1991;325(24):1696-1703.

[0583] 16.Fujiwara T, Konisbi M, Chida S, Okuyama K, Ogawa Y, TakecuchiY, et al. Surfactant replacement therapy with a single postventilatorydose of a reconstituted bovine surfactant in preterm neonates withrespiratory distress syndrome: final analysis of a multicenter,double-blind, randomized trial and comparison with similar trials.Pediatrics 1990;86(5):753-764.

[0584] 17.The OSIRIS Collaborative Group. Early versus delayed neonataladministration of a synthetic surfactant—the judgement of OSIRIS. Lancet1992;340(8832): 1363-1369.

[0585] 18.Ferrara T B, Hoekstra R E, Couser R I, Gaziano E P, Calvin SE, Payne N R, et al. Survival and follow-up of infants born at 23 to 26weeks of gestational age: effects of surfactant therapy. I Pediatr 1994;124(1): 119-124.

[0586] 19.Hallman M, Merritt T A, Jarvenpaa A-L, Boynton B, Mannino F,Gluck L, et al. Exogenous human surfactant for treatment of severerespiratory distress syndrome: a randomized prospective clinical trial.I Pediatr 1985; 106(6):963-969.

[0587] 20.Berry D D, Pramanik A K, Phillips J B ifi, Buchter D S,Kanarek K S, Easa D, et al. Comparison of the effect of three doses of asynthetic surfactant on the alveolar-arterial oxygen gradient in infantsweighing> or =1250 grams with respiratory distress syndrome. S Pediatr1994;124(2):294-301.

[0588] 21 .Gortner L, Bartmann P, Pohlandt F, Bernsau U, Porz F,Hellwege H, et al. Early treatment of respiratory distress syndrome withbovine surfactant in very preterm infants: a multicenter controlledclinical trial. Pediatr Pulmonol 1992;14(1):4-9.

[0589] 22.Corbet Al, Long W A, Murphy D J, Garcia-Prats I A, Lombardy LR, Wold D E. Reduced mortality in small premature infants treated atbirth with a single dose of synthetic surfactant. J Paediatr ChildHealth 1991 ;27(4):245-249.

[0590] 23.Bose C, Corbet A, Bose G, Garcia-Prats I, Lombardy L, Wold D,et al. Improved outcome at 28 days of age for very low birth weightinfants treated with a single dose of a synthetic surfactant. J Pediatr1990; 117:947-953.

[0591] 24.Corbet A, Bucciarelli R, Goldman S, Mammel M, Wold D, Long W,and the American Exosurf Pediatric Study Group. Decreased mortality rateamong small premature infants treated at birth with a single dose ofsynthetic surfactant: a multicenter controlled trial. I Pediatr1991;118(2):277-284.

[0592] 25.Merritt T A, Hahiman M, Bloom B T, Berry C, Benirschke K, SahnD, et al. Prophylactic treatment of very premature infants with humansurfactant. N Engl J Med 1986;315(13):785-790.

[0593] 26.Kendig 3W, Notter R H, Cox C, Reubens L I, Davis J M,Maniscalco W M, et al. A comparison of surfactant as immediateprophylaxis and as rescue therapy in newborns of less than 30 weeks'gestation. N Engl J Med 1991;324(13):865-871.

[0594] 27.Bgberts I, de Winter J P, Sedin G, deKleine M I, Broberger U,van Bel F, et al. Comparison of prophylaxis and rescue treatment withCurosurf in neonates less than 30 weeks' gestation: a randomized trial.Pediatrics 1993;92(6):768-774.

[0595] 28.Soll R F, Hoekstra R E, Fangman J J, Corbet A T, Adams 3M,James L S, et al. Multicenter trial of single-dose modified bovinesurfactant extract (Survanta) for prevention of respiratory distresssyndrome. Pediatrics 1990;85(6): 1092-1102.

[0596] 29.Heldt G P, Pesonen E, Merritt T A, Elias W, Sahn D I. Closureof the ductus arteriosus and mechanics of breathing in preterm infantsafter surfactant replacement therapy. Pediatr Res 1989;25(3):305-310.

[0597] 30.Hallman M, Merritt T A, Bry K, Berry C. Association betweenneonatal care practices and efficacy of exogenous human surfactant:results of a bicenter randomized trial. Pediatrics 1992;91(3):552-560.

[0598] 31.Lotze A, Knight G R, Martin G R, Bulas D I, Hull W M,O'Donnell R M, et al. Improved pulmonary outcome after exogenoussurfactant therapy for respiratory failure in term infants requiringextracorporeal membrane oxygenation. J Pediatr 1993;122(2):261-268.

[0599] 32.Raju T N, Langenberg P. Pulmonary hemorrhage and exogenoussurfactant therapy: a meta-analysis. J Pediatr 1993;123(4):603-610.

[0600] 33.Horbar I D, Soil R F, Sutherland 3M, Kotagal U, Philip A G S,Kessler D L, et al. A multicenter randomized, placebo-controlled trialof surfactant therapy for respiratory distress syndrome. N Engl J Med1989;320(15): 959-965.

[0601] 34.Horbar 3D, Wright I L, Soll R E, Fanaroff A A, Korones S B,Shankaran S, et al. A multicenter randomized trial comparing twosurfactants for the treatment of neonatal respiratory distress syndrome.J Pediatr 1993;123(5): 757-766.

[0602] 35.Hazan J, Chessex P, Piedboeuf B, Bourgeois M, Bard H, Long W.Energy expenditure during synthetic surfactant replacement therapy forneonatal respiratory distress syndrome. J Pediatr 1992;120(2, Part2):S29-S33.

[0603] 36.Speer C P, Harms K, Herting E, Neumann N, Curstedt T,Robertson B. Early versus late surfactant replacement therapy in severerespiratory distress syndrome. Lung 1990; 168(Suppl):870-876.

[0604] 37.Khammash H, Penman M, Wojtulewicz, J, Dunn M. Surfactanttherapy in full-term neonates with severe respiratory failure.Pediatrics 1993;92(1):135-139.

[0605] 38.Stevenson D, Waither F, Long W, Sell M, Paul T, Gong A, et al.Controlled trial of a single dose of synthetic surfactant at birth inpremature infants weighing 500 to 699 grams. J Pediatr 1992;120(2, Part2):S3-S12.

[0606] 39.Dunn M S, Shennan A T, Possmayer F. Single- versusmultiple-dose surfactant replacement therapy in neonates of 30 to 36weeks' gestation with respiratory distress syndrome. Pediatrics1990;86(4):564-571.

[0607] 40.Hellstrom-Westas L, Bell A R, Skov L, Greisen G, Svenningsen NW. Cerebroelectrical depression following surfactant treatment inpreterm neonates. Pediatrics 1992;89(4):643-647.

[0608] 41 .Zola E M, Gunkel I H, Chan R K, Lim M O, Knox I, Feldman B H,et al. Comparison of three dosing procedures for administration ofbovine surfactant to neonates with respiratory distress syndrome. JPediatr 1992;122(3):453-459.

[0609] 42.Annibale D I, Hulsey T C, Wallin L A, Engstrom P C. Clinicaldiagnosis and management of respiratory distress in preterm neonates:effect of participation in a controlled trial. Pediatrics1992;90(3):397-400.

[0610] 43.Goldman S L, Bosque E, McCann B, Lewis K. Pulmonary mechanicsin premature infants one month after treatment with syntheticsurfactant. J Pediatr 1992;120(2, Part 2):S25-S28.

[0611] 44.Speer C P, Robertson B, Curstedt T, Halhiday H L, CompagnoneD, Gefeller O, et al. Randomized European multicenter trial ofsurfactant replacement therapy for severe neonatal respiratory distresssyndrome: single versus multiple doses of Curosuil Pediatrics 1992;89(1):13-20.

[0612] 45.Gunkel J H, Banks P L. Surfactant therapy and intracranialhemorrhage: review of the literature and results of new analyses.Pediatrics 1993;92(6):775-786.

[0613] 46.Zola E M, Overbach A M, Gunkel 311, Mitchell B R, Nagle B T,DeMarco N G, et al. Treatment investigational new drug experience withSurvanta (beractant). Pediatrics 1993;91(3):546-551.

[0614] 47.van Houten J, Long W, Mullett M, Finer N, Derleth D, McMurrayB, et al. Pulmonary hemorrhage in premature infants after treatment withsynthetic surfactant: an autopsy evaluation. J Pediatr 1992; 120(2, Part2):S40-S44. Erratum appears in J Pediatr 1992;120(5):762.

[0615] 48.Rubin B K, Ramirez O, King M. Mucus rheology and transport inneonatal respiratory distress syndrome and the effect of surfactanttherapy. Chest 1992;101(4):1080-1085.

[0616] 49.Goldsmith L S, Greenspan J S, Rubenstein S D, Wolfson M R,Shaffer T H. Immediate improvement in lung volume after exogenoussurfactant: alveolar recruitment versus increased distention. J Pediatr1991;119(3):424-428.

[0617] 50.Bhutani V K, Abbasi S, Long W A, Gerdes I S. Pulmonarymechanics and energetics in preterm infants who had respiratory distresssyndrome treated with synthetic surfactant. J Pediatr 1992;120(2, Part2):S18-S24.

[0618] 51 .Bhat R, Dziedzic K, Bhutani V K, Vidyasagar D. Effect ofsingle dose surfactant on pulmonary function. Crit Care Med1990;18(6):590-595.

[0619] 52.Abbasi S, Bhutani V K, Gerdes I S. Long-term pulmonaryconsequences of respiratory distress syndrome in preterm infants treatedwith exogenous surfactant. J Pediatr 1993;122:446-452.

[0620] 53.Davis 3M, Veness-Meehan K, Notter R H, Bhutani V K, Kendig JW, Shapiro D L. Changes in pulmonary mechanics after the administrationof surfactant to infants with respiratory distress syndrome. N Engl JMed 1988;319(8):476-479.

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[0623] 56.Svenningsen N W, Bjorklund L, Vilstrup C, Werner 0. Lungmechanics (FRC and static pressure-volume diagram) after endotrachealsurfactant instillation: preliminary observations. Biol Neonate1992;61(Suppl 1):44-47.

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[0628] 61.Sitler C G, Turnage C S, McFadden B E, Smith E O, Adams 3M.Pump administration of exogenous surfactant: effects on oxygenation,heart rate, and chest wall movement of premature infants. J Perinatol1993;13(3):197-200.

[0629] 62.Centers for Disease Control. Update: Universal Precautions forprevention of transmission of human immunodeficiency virus, hepatitis Bvirus, and other bloodborne pathogens in health-care settings. MMWR1988;37:377-382,387-388.

[0630] Each of the applications and patents mentioned in this document,and each document cited or referenced in each of the these applicationsand patents, including during the prosecution of each of theapplications and patents (“application cited documents”) and anymanufacturer's instructions or catalogues for any products cited ormentioned in each of the applications and patents and in any of theapplication cited documents, are hereby incorporated herein byreference. Furthermore, all documents cited in this text, and alldocuments cited or referenced in documents cited in this text, and anymanufacturer's instructions or catalogues for any products cited ormentioned in this text, are hereby incorporated herein by reference.

[0631] Various modifications and variations of the described methods andsystem of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the claims.

1 8 1 177 PRT Homo sapiens MISC_FEATURE (2)..(2) Serine residue 2differs from the Homo sapiens sequence, in which this residue isProline. 1 Gly Ser Pro Gly Leu Lys Gly Asp Lys Gly Ile Pro Gly Asp LysGly 1 5 10 15 Ala Lys Gly Glu Ser Gly Leu Pro Asp Val Ala Ser Leu ArgGln Gln 20 25 30 Val Glu Ala Leu Gln Gly Gln Val Gln His Leu Gln Ala AlaPhe Ser 35 40 45 Gln Tyr Lys Lys Val Glu Leu Phe Pro Asn Gly Gln Ser ValGly Glu 50 55 60 Lys Ile Phe Lys Thr Ala Gly Phe Val Lys Pro Phe Thr GluAla Gln 65 70 75 80 Leu Leu Cys Thr Gln Ala Gly Gly Gln Leu Ala Ser ProArg Ser Ala 85 90 95 Ala Glu Asn Ala Ala Leu Gln Gln Leu Val Val Ala LysAsn Glu Ala 100 105 110 Ala Phe Leu Ser Met Thr Asp Ser Lys Thr Glu GlyLys Phe Thr Tyr 115 120 125 Pro Thr Gly Glu Ser Leu Val Tyr Ser Asn TrpAla Pro Gly Glu Pro 130 135 140 Asn Asp Asp Gly Gly Ser Glu Asp Cys ValGlu Ile Phe Thr Asn Gly 145 150 155 160 Lys Trp Asn Asp Arg Ala Cys GlyGlu Lys Arg Leu Val Val Cys Glu 165 170 175 Phe 2 534 DNA Homo sapiens 2ggaagcccgg gattgaaggg ggacaaaggc attcctggag acaaaggagc aaagggagaa 60agtgggcttc cagatgttgc ttctctgagg cagcaggttg aggccttaca gggacaagta 120cagcacctcc aggctgcttt ctctcagtat aagaaagttg agctcttccc aaatggccaa 180agtgtggggg agaagatttt caagacagca ggctttgtaa aaccatttac ggaggcacag 240ctgctgtgca cacaggctgg tggacagttg gcctctccac gctctgccgc tgagaatgcc 300gccttgcaac agctggtcgt agctaagaac gaggctgctt tcctgagcat gactgattcc 360aagacagagg gcaagttcac ctaccccaca ggagagtccc tggtctattc caactgggcc 420ccaggggagc ccaacgatga tggcgggtca gaggactgtg tggagatctt caccaatggc 480aagtggaatg acagggcttg tggagaaaag cgtcttgtgg tctgcgagtt ctga 534 3 1128DNA Homo sapiens 3 atgctgctct tcctcctctc tgcactggtc ctgctcacacagcccctggg ctacctggaa 60 gcagaaatga agacctactc ccacagaaca atgcccagtgcttgcaccct ggtcatgtgt 120 agctcagtgg agagtggcct gcctggtcgc gatggacgggatgggagaga gggccctcgg 180 ggcgagaagg gggacccagg tttgccagga gctgcagggcaagcagggat gcctggacaa 240 gctggcccag ttgggccaaa aggggacaat ggctctgttggagaacctgg accaaaggga 300 gacactgggc caagtggacc tccaggacct cccggtgtgcctggtccagc tggaagagaa 360 ggtgccctgg ggaagcaggg gaacatagga cctcagggcaagccaggccc aaaaggagaa 420 gctgggccta aaggagaagt aggtgcccca ggcatgcagggctcggcagg ggcaagaggc 480 ctcgcaggcc ctaagggaga gcgaggtgtc cctggtgagcgtggagtccc tggaaacaca 540 ggggcagcag ggtctgctgg agccatgggt ccccagggaagtccaggtgc caggggaccc 600 ccgggattga agggggacaa aggcattcct ggagacaaaggagcaaaggg agaaagtggg 660 cttccagatg ttgcttctct gaggcagcag gttgaggccttacagggaca agtacagcac 720 ctccaggctg ctttctctca gtataagaaa gttgagctcttcccaaatgg ccaaagtgtg 780 ggggagaaga ttttcaagac agcaggcttt gtaaaaccatttacggaggc acagctgctg 840 tgcacacagg ctggtggaca gttggcctct ccacgctctgccgctgagaa tgccgccttg 900 caacagctgg tcgtagctaa gaacgaggct gctttcctgagcatgactga ttccaagaca 960 gagggcaagt tcacctaccc cacaggagag tccctggtctattccaactg ggccccaggg 1020 gagcccaacg atgatggcgg gtcagaggac tgtgtggagatcttcaccaa tggcaagtgg 1080 aatgacaggg cttgtggaga aaagcgtctt gtggtctgcgagttctga 1128 4 375 PRT Homo sapiens 4 Met Leu Leu Phe Leu Leu Ser AlaLeu Val Leu Leu Thr Gln Pro Leu 1 5 10 15 Gly Tyr Leu Glu Ala Glu MetLys Thr Tyr Ser His Arg Thr Met Pro 20 25 30 Ser Ala Cys Thr Leu Val MetCys Ser Ser Val Glu Ser Gly Leu Pro 35 40 45 Gly Arg Asp Gly Arg Asp GlyArg Glu Gly Pro Arg Gly Glu Lys Gly 50 55 60 Asp Pro Gly Leu Pro Gly AlaAla Gly Gln Ala Gly Met Pro Gly Gln 65 70 75 80 Ala Gly Pro Val Gly ProLys Gly Asp Asn Gly Ser Val Gly Glu Pro 85 90 95 Gly Pro Lys Gly Asp ThrGly Pro Ser Gly Pro Pro Gly Pro Pro Gly 100 105 110 Val Pro Gly Pro AlaGly Arg Glu Gly Ala Leu Gly Lys Gln Gly Asn 115 120 125 Ile Gly Pro GlnGly Lys Pro Gly Pro Lys Gly Glu Ala Gly Pro Lys 130 135 140 Gly Glu ValGly Ala Pro Gly Met Gln Gly Ser Ala Gly Ala Arg Gly 145 150 155 160 LeuAla Gly Pro Lys Gly Glu Arg Gly Val Pro Gly Glu Arg Gly Val 165 170 175Pro Gly Asn Thr Gly Ala Ala Gly Ser Ala Gly Ala Met Gly Pro Gln 180 185190 Gly Ser Pro Gly Ala Arg Gly Pro Pro Gly Leu Lys Gly Asp Lys Gly 195200 205 Ile Pro Gly Asp Lys Gly Ala Lys Gly Glu Ser Gly Leu Pro Asp Val210 215 220 Ala Ser Leu Arg Gln Gln Val Glu Ala Leu Gln Gly Gln Val GlnHis 225 230 235 240 Leu Gln Ala Ala Phe Ser Gln Tyr Lys Lys Val Glu LeuPhe Pro Asn 245 250 255 Gly Gln Ser Val Gly Glu Lys Ile Phe Lys Thr AlaGly Phe Val Lys 260 265 270 Pro Phe Thr Glu Ala Gln Leu Leu Cys Thr GlnAla Gly Gly Gln Leu 275 280 285 Ala Ser Pro Arg Ser Ala Ala Glu Asn AlaAla Leu Gln Gln Leu Val 290 295 300 Val Ala Lys Asn Glu Ala Ala Phe LeuSer Met Thr Asp Ser Lys Thr 305 310 315 320 Glu Gly Lys Phe Thr Tyr ProThr Gly Glu Ser Leu Val Tyr Ser Asn 325 330 335 Trp Ala Pro Gly Glu ProAsn Asp Asp Gly Gly Ser Glu Asp Cys Val 340 345 350 Glu Ile Phe Thr AsnGly Lys Trp Asn Asp Arg Ala Cys Gly Glu Lys 355 360 365 Arg Leu Val ValCys Glu Phe 370 375 5 125 PRT Homo sapiens 5 Val Glu Leu Phe Pro Asn GlyGln Ser Val Gly Glu Lys Ile Phe Lys 1 5 10 15 Thr Ala Gly Phe Val LysPro Phe Thr Glu Ala Gln Leu Leu Cys Thr 20 25 30 Gln Ala Gly Gly Gln LeuAla Ser Pro Arg Ser Ala Ala Glu Asn Ala 35 40 45 Ala Leu Gln Gln Leu ValVal Ala Lys Asn Glu Ala Ala Phe Leu Ser 50 55 60 Met Thr Asp Ser Lys ThrGlu Gly Lys Phe Thr Tyr Pro Thr Gly Glu 65 70 75 80 Ser Leu Val Tyr SerAsn Trp Ala Pro Gly Glu Pro Asn Asp Asp Gly 85 90 95 Gly Ser Glu Asp CysVal Glu Ile Phe Thr Asn Gly Lys Trp Asn Asp 100 105 110 Arg Ala Cys GlyGlu Lys Arg Leu Val Val Cys Glu Phe 115 120 125 6 28 PRT Homo sapiens 6Asp Val Ala Ser Leu Arg Gln Gln Val Glu Ala Leu Gln Gly Gln Val 1 5 1015 Gln His Leu Gln Ala Ala Phe Ser Gln Tyr Lys Lys 20 25 7 24 PRTArtificial Sequence Preferred amino terminal sequence of rSPD(n/CRD)polypeptide. 7 Gly Ser Pro Gly Leu Lys Gly Asp Lys Gly Ile Pro Gly AspLys Gly 1 5 10 15 Ala Lys Gly Glu Ser Gly Leu Pro 20 8 12 PRTAspergillus fumigatus 8 Ala Thr Trp Thr Cys Ile Asn Gln Gln Leu Asn Pro1 5 10

1. A recombinant polypeptide comprising amino acids 54-177 of SEQ ID NO:1 or a fragment thereof that retains one or more biological activitiesof an rSPD(n/CRD) polypeptide.
 2. An isolated rSPD(n/CRD) polypeptidecomprising amino acids 1-177 of SEQ ID NO: 1 or 179-355 of SEQ ID NO: 4or a fragment thereof that retains one or more biological activities ofthe polypeptide of SEQ ID NO: 1 or SEQ ID NO:
 4. 3. The polypeptide ofclaim 1 that further comprises the sequence of amino acids 25-53 of SEQID NO: 1, located immediately N-terminal to said amino acids 54-177 ofSEQ ID NO:
 1. 4. The polypeptide of claim 3 which comprises aconservative amino acid substitution relative to the sequence of aminoacids 25-53 of SEQ ID NO:
 1. 5. The recombinant polypeptide of claims 1or 2 which comprises a conservative amino acid substitution relative tothe sequence of amino acids 54-177 of SEQ ID NO:
 1. 6. The polypeptideof claim 1 that further comprises at least one G-Xaa-Yaa sequencelocated N-terminal to said polypeptide of claim
 1. 7. The polypeptide ofclaim 6 which comprises a contiguous stretch of 8 of said G-Xaa-Yaasequences.
 8. The polypeptide of claim 7 wherein said contiguous stretchof 8 G-Xaa-Yaa sequences consists of amino acids 1-24 of SEQ ID NO: 1.9. The polypeptide of claim 3 which further comprises at least oneG-Xaa-Yaa sequence located immediately N-terminal to said amino acids25-53 of SEQ ID NO:
 1. 10. The polypeptide of claim 9 which comprises astretch of 8 contiguous G-Xaa-Yaa sequences located immediatelyN-terminal to said amino acids 25-53 of SEQ ID NO:
 1. 11. Thepolypeptide of claim 10 which comprises a conservative amino acidsubstitution relative to the sequence of SEQ ID NO:
 1. 12. A polypeptidecomprising the amino acid sequence of SEQ ID NO:
 1. 13. A polypeptideconsisting of the amino acid sequence of SEQ ID NO:
 1. 14. A method oftreatment or prophylaxis of a disease or disorder, the method comprisingadministering a therapeutically or prophylactically effective amount ofan rSPD(n/CRD) polypeptide to an individual in need of such treatment orprophylaxis.
 15. The method of claim 14 wherein said rSPD(n/CRD)polypeptide comprises a polypeptide according to claim 1 or claim
 5. 16.The method of claim 14 wherein said disease or disorder comprises aninflammatory disease.
 17. The method of claim 16 wherein saidinflammatory disease is selected from the group consisting of allergyand inflammatory lung disease.
 18. The method of claim 17 wherein saidallergy is an allergy to the house dust mite (Dermatophagoides spp), afungus or fungal spores.
 19. The method of claim 18 wherein said fungusis Aspergillus fumigatus.
 20. The method of claim 14 wherein saiddisease or disorder is associated with microbial infection.
 21. Themethod of claim 20 wherein said microbial infection is a bacterial orviral infection.
 22. The method of claim 20 wherein said microbialinfection is an infection of the lung.
 23. A method of reducing airwayhyperresponsiveness, serum IgE levels or eosinophilia in an individual,the method comprising administering to the individual an rSPD(n/CRD)polypeptide.
 24. The method of claim 23 wherein said rSPD(n/CRD)polypeptide comprises a polypeptide of claim 1 or claim
 5. 25. A methodof reducing alveolar macrophage number in an individual, the methodcomprising administering to the individual an rSPD(n/CRD) polypeptide.26. The method of claim 25 wherein said rSPD(n/CRD) polypeptidecomprises a polypeptide of claim 1 or claim
 5. 27. The method of claim25 wherein said method enhances the clearance of apoptotic alveolarmacrophages, or enhances the clearance of necrotic alveolar macrophages,or both.
 28. The method of claim 14 wherein said method comprisessurfactant replacement therapy.
 29. An isolated polynucleotide encodingan rSPD(n/CRD) polypeptide.
 30. A vector comprising a polynucleotidesequence encoding an rSPD(n/CRD) polypeptide.
 31. The vector of claim 30which comprises the sequence of SEQ ID NO:
 2. 32. A host cell comprisingthe vector of claim
 30. 33. The host cell of claim 32 which comprises avector which comprises the sequence of SEQ ID NO:
 2. 34. Thepolynucleotide of claim 29, wherein said polynucleotide comprises thesequence of nucleotides 157-534 of SEQ ID NO:
 2. 35. The polynucleotideof claim 34, wherein said polynucleotide comprises a mutation thatresults in a conservative amino acid substitution in the polypeptideencoded by said nucleotides 157-534 of SEQ ID NO: 2, wherein saidencoded polypeptide retains one or more biological activities of anrSPD(n/CRD) polypeptide.
 36. The polynucleotide of claim 29, whereinsaid polynucleotide comprises the sequence of nucleotides 73-534 of SEQID NO:
 2. 37. The polynucleotide of claim 29, wherein saidpolynucleotide comprises the sequence of nucleotides 1-534 of SEQ ID NO:2.
 38. The polynucleotide of claim 36 or 37 which comprises a mutationthat results in a conservative amino acid substitution in thepolypeptide encoded by said nucleotides 1-534 of SEQ ID NO: 2, whereinsaid encoded polypeptide retains one or more biological activities of anrSPD(n/CRD) polypeptide.
 39. An isolated polynucleotide comprising thesequence of nucleotides 157-534 of SEQ ID NO:
 2. 40. The polynucleotideof claim 39 which comprises a mutation that results in a conservativeamino acid substitution in the polypeptide encoded by said nucleotides157-534 of SEQ ID NO: 2, wherein said encoded polypeptide retains one ormore biological activities of an rSPD(n/CRD) polypeptide.
 41. Anisolated polynucleotide which comprises the sequence of nucleotides73-534 of SEQ ID NO:
 2. 42. The polynucleotide of claim 41 whichcomprises a mutation that results in a conservative amino acidsubstitution in the polypeptide encoded by said nucleotides 73-534 ofSEQ ID NO: 2, wherein said encoded polypeptide retains one or morebiological activities of an rSPD(n/CRD) polypeptide.
 43. An isolatedpolynucleotide which comprises the sequence of nucleotides 1-534 of SEQID NO:
 2. 44. The polynucleotide of claim 43 which comprises a mutationthat results in a conservative amino acid substitution in thepolypeptide encoded by said nucleotides 1-534 of SEQ ID NO: 2, whereinsaid encoded polypeptide retains one or more biological activities of anrSPD(n/CRD) polypeptide.
 45. A composition comprising a recombinantpolypeptide of claim 1 or claim 5 and a pharmaceutically acceptablecarrier.
 46. A composition comprising a recombinant polypeptide of claim1 or claim 5, or a polynucleotide of claim 29, or a vector of claim 30or a host cell of claim 32 and one or more of the following components:a lung extract, a phospholipid, a fatty acid, a surfactant-associatedprotein, a cetyl alcohol, a long-chain repeating alcohol, tyloxapol,colfosceril palmitate (dipalmitoylphosphatidylcholine), palmitic acid,tripalmitin, poractant alfa and sodium chloride.
 47. The composition ofclaim 46 further comprising a pharmaceutically acceptable carrier. 48.The composition of claim 46 wherein said lung extract comprises a bovinelung extract.
 49. The composition of claim 48 wherein said bovine lungextract comprises a calf lung extract.
 50. The composition of claim 46wherein said phospholipid comprises a phosphatidylcholine or adisaturated phosphatidylcholine.
 51. The composition of claim 46 whereinsaid surfactant-associated protein comprises a surfactant-associatedprotein B or a surfactant-associated protein C.
 52. A compositioncomprising a polynucleotide of claim 29, a vector of claim 30 or a hostcell of claim 32 and a pharmaceutically acceptable carrier.
 53. A methodof identifying a molecule which binds to an rSPD(n/CRD) polypeptide, themethod comprising contacting an rSPD(n/CRD) polypeptide to a candidatemolecule and detecting whether the candidate molecule binds to therSPD(n/CRD) polypeptide.
 54. A method of identifying an agonist orantagonist of an rSPD(n/CRD) polypeptide, the method comprising: (a)providing a cell or organism; (b) contacting the cell or organism withan rSPD(n/CRD) polypeptide; (c) contacting the cell with a candidatemolecule; and (d) detecting a change in a biological activity of saidrSPD(n/CRD), wherein said change is indicative that said candidatemolecule is an agonist or antagonist of said rSPD(n/CRD) polypeptide.55. The method of claim 54, in which said biological activity of saidrSPD(n/CRD) is selected from the group consisting of: reduction ofperipheral blood eosinophilia, reduction of serum IgE levels, reductionof serum IgE levels, reduction in airway hyperresponsiveness, reductionin alveolar macrophage number, reduction of phospholipid levels inlavage, down-regulation of Eotaxin expression, reduction in MCP-1expression, down-regulation of MIP-1α expression, and down-regulation ofMIP-2 expression.
 56. A method for the treatment of a conditioninvolving the production of free DNA on the surface of cells, saidmethod comprising administering a therapeutically effective amount of anSP-D polypeptide to an individual in need of such treatment.
 57. Themethod of claim 56 wherein said SP-D polypeptide binds to nucleic acidvia its carbohydrate-binding domain.
 58. A method for the treatment of acondition involving the production of free DNA on the surface of cells,said method comprising administering a therapeutically effective amountof an rSPD(n/CRD) polypeptide to an individual in need of suchtreatment.
 59. A method for the treatment of a condition involving theaccumulation of free nucleic acid in the lung, said method comprisingadministering a therapeutically effective amount of an SP-D polypeptideto an individual in need of such treatment.
 60. A method for thetreatment of a condition involving the accumulation of free nucleic acidin the lung, said method comprising administering a therapeuticallyeffective amount of an rSPD(n/CRD) polypeptide to an individual in needof such treatment.
 61. A method for the treatment of cystic fibrosis,said method comprising administering a therapeutically effective amountof an SP-D polypeptide to an individual in need of such treatment.
 62. Amethod for the treatment of cyctic fibrosis, said method comprisingadministering a therapeutically effective amount of an rSPD(n/CRD)polypeptide to an individual in need of such treatment.
 63. A method forthe clearance of necrotic and apoptotic cells, said method comprisingadministering a therapeutically effective amount of an SP-D polypeptideto an individual in need of such clearance.
 64. A method for theclearance of necrotic and apoptotic cells, said method comprisingadministering a therapeutically effective amount of an rSPD(n/CRD)polypeptide to an individual in need of such clearance.
 65. The methodof claim 63 or claim 64 wherein said necrotic or apoptotic cells arepresent in the lung.
 66. A method for the reduction of autoantibodyproduction in autoimmune disease, the method comprising administering atherapeutically effective amount of an SP-D polypeptide to an individualin need of such reduction.
 67. A method for the reduction ofautoantibody production in autoimmune disease, the method comprisingadministering a therapeutically effective amount of an rSPD(n/CRD)polypeptide to an individual in need of such reduction.
 68. A method fortreating an autoimmune disease comprising administering to a patient inneed of such treatment a pharmaceutically effective amount of an SP-Dpolypeptide.
 69. A method for treating an autoimmune disease comprisingadministering to a patient in need of such treatment a pharmaceuticallyeffective amount of an rSPD(n/CRD) polypeptide.